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Abstract:

The invention generally features compositions and methods based on the
structure-based design of alpha 1-3 N-Acetylgalactosaminyltransferase
(alpha 3 GaINAc-T) enzymes from alpha 1-3 galactosyltransferase (a3Gal-T)
that can transfer 2'-modified galactose from the corresponding
UDP-derivatives due to substitutions that broaden the alpha 3Gal-T donor
specificity and make the enzyme a3 GaINAc-T.

Claims:

1. A polypeptide fragment of an alpha 1,3
N-acetylgalactosaminyltransferase (alpha 3GalNaCT) that retains the
ability to transfer a sugar with a chemically reactive functional group
from a sugar donor to a sugar acceptor.

3. The polypeptide fragment of claim 2, wherein the polypeptide fragment
comprises one or more substitutions in the donor substrate binding site.

4. The polypeptide fragment of claim 2, wherein the polypeptide fragment
comprises one or more substitutions in the hinge region.

5. The polypeptide fragment of claim 2, wherein the polypeptide fragment
comprises one or more substitutions near the DXD motif.

6. The polypeptide fragment of claim 3, wherein the one or more
substitutions in the substrate binding site comprise an amino acid
substitution at position 280, 281, or 282 corresponding to bovine alpha
1,3 galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21).

7. The polypeptide fragment of claim 4, wherein the one or more
substitutions in the hinge region comprise an amino acid substitution at
position 191 corresponding to bovine alpha 1,3 galactosyltransferase
(alpha 3 Gal-T) (SEQ ID NO: 21).

8. The polypeptide fragment of claim 5, wherein the one or more
substitutions close to the DXD motif comprise an amino acid substitution
at position 228 corresponding to bovine alpha 1,3 galactosyltransferase
(alpha 3 Gal-T) (SEQ ID NO: 21)

9. The polypeptide fragment of claim 6, wherein a serine (S) is
substituted for a histidine (H) at amino acid position 280, a glycine (G)
is substituted for an alanine (A) at amino acid position 281, or a
glycine (G) is substituted for an alanine at amino acid position 282 of
(SEQ ID NO 21).

10. The polypeptide fragment of claim 7, wherein a serine (S) or an
alanine (A) is substituted for a proline (P) at amino acid position 191
corresponding to (SEQ ID NO: 21).

11. The polypeptide fragment of claim 8, wherein a glutamine (Q) is
replaced with a methionine (M) at amino acid position 228 of (SEQ ID
NO:21).

12. A polypeptide fragment of an alpha 1,3
N-acetylgalactosaminyltransferase (alpha3GalNAc-T) that transfers a sugar
with a chemically reactive functional group from a sugar donor to a sugar
acceptor, wherein the polypeptide fragment comprises and one of SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO; 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20 or
a polypeptide fragment of an alpha 1,3 N-acetylgalactosaminyltransferase
(alpha3GalNac-T) that retains that ability to transfer a sugar with a
chemically reactive functional group from a sugar donor to a sugar
acceptor and catalyzes the formation of an oligosaccharide.

70. A composition comprising a polypeptide fragment of an alpha 1,3
N-acetylgalactosaminytransferase (alpha 3GalNaCT) that retains the
ability to transfer a sugar with a chemically reactive functional group
from a sugar donor to a sugar acceptor, or a composition comprising a
polypeptide fragment of an alpha 1,3 N-acetylgalactosaminyltransferase
(alpha3GalNAc-T) that transfers a sugar with a chemically reactive
functional group from a sugar donor to a sugar acceptor, wherein the
polypeptide fragment comprises and one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO; 12, SEQ ID NO: 14, SEQ
ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, or a composition comprising a
polypeptide fragment of an alpha 1,3 N-acetylgalactosaminyltransferase
(alpha3GalNAc-T) that retains that ability to transfer a sugar with a
chemically reactive functional group from a sugar donor to a sugar
acceptor and catalyzes the formation of an oligosaccharide.

71-91. (canceled)

92. An immunological composition comprising a polypeptide fragment of an
alpha 1,3 N-acetylgalactosaminyltransferase (alpha 3GalNAc-T) that
retains the ability to transfer a sugar with a chemically reactive
functional group from a sugar donor to a sugar acceptor, or an
immunological composition comprising a polypeptide fragment of an alpha
1,3 N-acetylgalactosaminyltransferase (alpha3GalNAc-T) that transfers a
sugar with a chemically reactive functional group from a sugar donor to a
sugar acceptor, wherein the polypeptide fragment comprises and one of SEQ
ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO; 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20,
and wherein one or more antibodies are conjugated to the chemically
reactive functional group, or an immunological composition comprising a
polypeptide fragment of an alpha 1,3 N-acetylgalactosaminyltransferase
(alpha3GalNAc-T) that retains that ability to transfer a sugar with a
chemically reactive functional group from a sugar donor to a sugar
acceptor and catalyzes the formation of an oligosaccharide, and wherein
one or more antibodies are conjugated to the chemically reactive
functional group.

93-113. (canceled)

114. A method of coupling an agent to a carrier protein comprising:
incubating a reaction mixture comprising a polypeptide fragment of an
alpha 1,3 N-acetylgalactosaminyltransferase (alpha 3GalNAc T) that
retains the ability to transfer a sugar with a chemically reactive
functional group from a sugar donor to a sugar acceptor, wherein the
sugar donor is coupled to an agent and the sugar acceptor is a carrier
protein.

115-137. (canceled)

138. A method for the diagnosis or treatment of a subject suffering from
a disease or disorder comprising: administering to the subject an
effective amount of an isolated glycoprotein synthesized by the method
according to claim 60, wherein one or more agents are linked to the sugar
donor, thereby diagnosing or treating a subject suffering from a disease
or disorder.

139-140. (canceled)

141. A method for imaging a target cell or tissue comprising:
administering to a subject an oligosaccharide synthesized by the method
according to claim 48, and wherein one or more imaging agents are linked
to the sugar donor, thereby imaging a target cell or tissue.

142. A method for synthesizing a detectable Gal beta 1-4GlcNAc epitope
comprising: synthesizing an oligosaccharide according to the method of
claim 50, wherein the sugar donor comprises a 2' modified Gal residue and
wherein or more detection agents are linked to the 2' modified Gal
residue thereby synthesizing a detectable Gal beta 1-4GlcNAc epitope.

143-145. (canceled)

146. A kit comprising packaging material, and polypeptide fragment from
an alpha 1,3 N-acetylgalactosaminyltransferase (alpha3GalNac-T) according
to claim 1.

147-150. (canceled)

Description:

FIELD OF THE INVENTION

[0002] The invention relates generally to the structure-based design of
alpha 1-3 N-Acetylgalactosaminyltransferase (alpha 3 GalNAc-T) enzymes
from alpha 1-3galactosyltransferase (a3Gal-T). The novel alpha 1-3
GalNAc-transferases described herein can transfer 2'-modified galactose
from the corresponding UDP-derivatives due to substitutions that broaden
the alpha 3Gal-T donor specificity and make the enzyme a3 GalNAc-T.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to the field of glycobiology, and
specifically to glycosyltransferases, a superfamily of enzymes that are
involved in synthesizing the carbohydrate moieties of glycoproteins,
glycolipids and glycosaminoglycans. The present invention provides the
structure-based design of novel glycosyltransferases and their biological
applications.

[0004] Glycans can be classified as linear or branched sugars. The linear
sugars are the glycosaminoglycans comprising polymers of sulfated
disaccharide repeat units that are O-linked to a core protein, forming a
proteoglycan aggregate (Raman et al. 2005). The branched glycans are
found as N-linked and O-linked sugars on glycoproteins or on glycolipids
(Lowe et al., 2003). These carbohydrate moieties of the linear and
branched glycans are synthesized by a super family of enzymes, the
glycosyltransferases (GTs), which transfer a sugar moiety from a sugar
donor to an acceptor molecule. Although GTs catalyze chemically similar
reactions in which a monosaccharide is transferred from an activated
derivative, such as a UDP-sugar, to an acceptor, very few GTs bear
similarity in primary structure.

[0005] Eukaryotic cells express several classes of oligosaccharides
attached to proteins or lipids. Animal glycans can be N-linked via
beta-GlcNAc to Asn (N-glycans), O-linked via -GalNAc to Ser/Thr
(O-glycans), or can connect the carboxyl end of a protein to a
phosphatidylinositol unit (GPI-anchors) via a common core glycan
structure.

[0006] The structural information of glycosyltransferases has revealed
that the specificity of the sugar donor in these enzymes is determined by
a few residues in the sugar-nucleotide binding pocket of the enzyme,
which is conserved among the family members from different species. This
conservation has made it possible to reengineer the existing
glycosyltransferases with broader sugar donor specificities. Mutation of
these residues generates novel glycosyltransferases that can transfer a
sugar residue with a chemically reactive functional group to
N-acetylglucosamine (GlcNAc), galactose (Gal) and xylose residues of
glycoproteins, glycolipids and proteoglycans (glycoconjugates). Thus,
there is potential to develop mutant glycosyltransferases to produce
glycoconjugates carrying sugar moieties with reactive groups that can be
used in the assembly of bio-nanoparticles to develop targeted-drug
delivery systems or contrast agents for medical uses.

[0007] Accordingly, methods to synthesize N-acetylglucosamine linkages
have many applications in research and medicine, including in the
development of pharmaceutical agents and improved vaccines that can be
used to treat disease.

SUMMARY OF THE INVENTION

[0008] As described below, the present invention features the
structure-based design of alpha 1-3 N-Acetylgalactosaminyltransferase
(alpha 3 GalNAc-T) enzymes from alpha 1-3galactosyltransferase (a3Gal-T).
The novel alpha 1-3 GalNAc-transferases described herein can transfer
2'-modified galactose from the corresponding UDP-derivatives due to
substitutions that broaden the alpha 3Gal-T donor specificity and make
the enzyme a3 GalNAc-T.

[0009] In one aspect the invention provides a polypeptide fragment of an
alpha 1,3 N-acetylgalactosaminyltransferase (alpha 3GalNaCT) that retains
the ability to transfer a sugar with a chemically reactive functional
group from a sugar donor to a sugar acceptor.

[0010] In one embodiment, the polypeptide fragment comprises a donor
substrate-binding site, a hinge region and a DXD motif. In another
embodiment the polypeptide fragment comprises one or more substitutions
in the donor substrate-binding site.

[0011] In a further embodiment, the polypeptide fragment comprises one or
more substitutions in the hinge region. In still another embodiment,
polypeptide fragment comprises one or more substitutions near the DXD
motif.

[0012] In a related embodiment, the one or more substitutions in the
substrate binding site comprise an amino acid substitution at position
280, 281, or 282 corresponding to bovine alpha 1,3 galactosyltransferase
(alpha 3 Gal-T) (SEQ ID NO: 21). In another related embodiment, the one
or more substitutions in the substrate hinge region comprise an amino
acid substitution at position 191 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In one embodiment,
the one or more substitutions close to the DXD motif comprise an amino
acid substitution at position 228 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21). In another
embodiment, wherein a serine (S) is substituted for a histidine (H) at
amino acid position 280, a glycine (G) is substituted for an alanine (A)
at amino acid position 281, or a glycine (G) is substituted for an
alanine at amino acid position 282 of (SEQ ID NO 21). In another
embodiment, a serine (S) or an alanine (A) is substituted for a proline
(P) at amino acid position 191 corresponding to (SEQ ID NO: 21). In still
another embodiment, a glutamine (Q) is replaced with a a methionine (M)
at amino acid position 228 of (SEQ ID NO:21).

[0014] In one embodiment of the above-mentioned aspects, the sugar
acceptor is selected from the group consisting of galactose beta 1,4
GlcNac and galactose beta 1,4 glucose.

[0015] In another embodiment of the above-mentioned aspects, the sugar
with a chemically reactive functional group is selected from the group
consisting of UDP-GalNAc, UDP-galactose, and UDP-galactose analogues.

[0016] In one embodiment, the UDP-galactose analogue comprises an azido
group, a keto group or a thiol group.

[0017] In another embodiment, the azido group, the keto group or the thiol
group is substituted at the C2 position of galactose. In a further
embodiment, the one or more agents are linked to a sugar moiety of the
sugar donor. In one embodiment, the one or more agents is selected from
the group consisting of: single chain antibodies, bacterial toxins,
growth factors, therapeutic agents, targeting agents, contrast agents,
chemical labels, radiolabels, and fluorescent labels.

[0018] In another aspect, the invention features a polypeptide fragment of
an alpha 1,3 N-Acetylgalactosaminyltransferase (alpha3GalNac-T) that
retains that ability to transfer a sugar with a chemically reactive
functional group from a sugar donor to a sugar acceptor and catalyzes the
formation of an oligosaccharide. In one embodiment, the oligosaccharide
is a disaccharide or a trisaccharide. In another embodiment, the
trisaccharide is selected from the group consisting of: GalNAc alpha
1-3Galbeta 1-4Gal, GalNAc alpha 1-3-Galbeta 1-4GlcNAc, 2'-modified-Gal
alpha 1-3 Gal beta 1-4Gal or 2'-modified-Galalpha 1-3-Gal beta 1-4GlcNAc.
In one embodiment, the polypeptide fragment comprises any one of SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO; 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

[0021] In one embodiment, the expression cassette or vector comprises the
nucleic acid of the aspects as described herein.

[0022] In another aspect, the invention features an expression cassette or
vector comprising a nucleic acid segment encoding a polypeptide fragment
from an alpha 1,3 N acetylgalactosaminyltransferase (alpha 3Gal NAcT that
transfers a sugar with a chemically reactive functional group from a
sugar donor to a sugar acceptor.

[0023] In one embodiment, the cell comprises the expression cassette or
vector as described in the aspects herein.

[0024] In another aspect, the invention features a method to of making an
oligosaccharide comprising incubating a reaction mixture comprising a
polypeptide fragment of an alpha 1,3 N-acetylgalactosaminyltransferase
(alpha 3GalNaCT) that retains the ability to transfer a sugar with a
chemically reactive functional group with a sugar donor and a sugar
acceptor.

[0025] In one embodiment, the polypeptide fragment comprises a donor
substrate binding site, a hinge region and a DXD motif. In another
embodiment, the polypeptide fragment comprises one or more substitutions
in the donor substrate binding site. In another embodiment, the
polypeptide fragment comprises one or more substitutions in the hinge
region. In another embodiment, the polypeptide fragment comprises one or
more substitutions near the DXD motif.

[0026] In a related embodiment, the one or more substitutions in the
substrate binding site comprise an amino acid substitution at position
280, 281, or 282 corresponding to bovine alpha 1,3 galactosyltransferase
(alpha 3 Gal-T) (SEQ ID NO: 21). In another related embodiment, the one
or more substitutions in the substrate hinge region comprise an amino
acid substitution at position 191 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In one embodiment,
the one or more substitutions close to the DXD motif comprise an amino
acid substitution at position 228 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21). In another
embodiment, wherein a serine (S) is substituted for a histidine (H) at
amino acid position 280, a glycine (G) is substituted for an alanine (A)
at amino acid position 281, or a glycine (G) is substituted for an
alanine at amino acid position 282 of (SEQ ID NO 21). In another
embodiment, a serine (S) or an alanine (A) is substituted for a proline
(P) at amino acid position 191 corresponding to (SEQ ID NO: 21). In still
another embodiment, a glutamine (Q) is replaced with a methionine (M) at
amino acid position 228 of (SEQ ID NO:21).

[0028] In one embodiment, the sugar acceptor is selected from the group
consisting of galactose beta 1,4 GlcNac and galactose beta 1,4 glucose.

[0029] In another embodiment, the sugar with a chemically reactive
functional group is selected from the group consisting of UDP-GalNAc,
UDP-galactose, and UDP-galactose analogues.

[0030] In a further embodiment, the UDP-galactose analogue comprises an
azido group, a keto group or a thiol group. In another embodiment, the
azido group, the keto group or the thiol group is substituted at the C2
position of galactose. In still a further embodiment, one or more agents
are linked to a sugar moiety of the sugar donor.

[0031] In one embodiment, the one or more agents is selected from the
group consisting of: single chain antibodies, bacterial toxins, growth
factors, therapeutic agents, targeting agents, contrast agents, chemical
labels, radiolabels, and fluorescent labels.

[0032] In another embodiment, the oligosaccharide is a disaccharide or a
trisaccharide. In a related embodiment, the trisaccharide is selected
from the group consisting of: GalNAc alpha1-3Galbeta 1-4Gal, GalNAc
alpha1-3-Galbeta 1-4GlcNAc, 2'-modified-Gal alpha 1-3 Gal beta 1-4Gal or
2'-modified-Galalpha 1-3-Gal beta 1-4GlcNAc.

[0034] In another aspect, the invention features an oligosaccharide
synthesized by the method comprising incubating a reaction mixture
comprising a polypeptide fragment of an alpha 1,3
N-acetylgalactosylaminotransferase (alpha 3GalNAc T) that retains the
ability to transfer a sugar with a chemically reactive functional group
with a sugar donor and a sugar acceptor.

[0035] In one embodiment, the polypeptide fragment comprises a donor
substrate binding site, a hinge region and a DXD motif. In another
embodiment, the polypeptide fragment comprises one or more substitutions
in the donor substrate binding site. In one embodiment, the polypeptide
fragment comprises one or more substitutions in the hinge region. In one
embodiment, the polypeptide fragment comprises one or more substitutions
near the DXD motif.

[0036] In a related embodiment, the one or more substitutions in the
substrate binding site comprise an amino acid substitution at position
280, 281, or 282 corresponding to bovine alpha 1,3 galactosyltransferase
(alpha 3 Gal-T) (SEQ ID NO: 21). In another related embodiment, the one
or more substitutions in the substrate hinge region comprise an amino
acid substitution at position 191 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In one embodiment,
the one or more substitutions close to the DXD motif comprise an amino
acid substitution at position 228 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21). In another
embodiment, wherein a serine (S) is substituted for a histidine (H) at
amino acid position 280, a glycine (G) is substituted for an alanine (A)
at amino acid position 281, or a glycine (G) is substituted for an
alanine at amino acid position 282 of (SEQ ID NO 21). In another
embodiment, a serine (S) or an alanine (A) is substituted for a proline
(P) at amino acid position 191 corresponding to (SEQ ID NO: 21). In still
another embodiment, a glutamine (Q) is replaced with a methionine (M) at
amino acid position 228 of (SEQ ID NO:21).

[0038] In one embodiment, the sugar acceptor is selected from the group
consisting of galactose beta 1,4 GlcNAc and galactose beta 1,4 glucose.
In another embodiment, the sugar with a chemically reactive functional
group is selected from the group consisting of UDP-GalNAc, UDP-galactose,
and UDP-galactose analogues.

[0039] In another embodiment, the UDP-galactose analogue comprises an
azido group, a keto group or a thiol group. In a related embodiment, the
azido group, the keto group or the thiol group is substituted at the C2
position of galactose.

[0040] In a related embodiment, one or more agents are linked to a sugar
moiety of the sugar donor. In another embodiment, the one or more agents
is selected from the group consisting of: single chain antibodies,
bacterial toxins, growth factors, therapeutic agents, targeting agents,
contrast agents, chemical labels, radiolabels, and fluorescent labels.

[0041] In one embodiment, the oligosaccharide is a disaccharide or a
trisaccharide. In another embodiment, the trisaccharide is selected from
the group consisting of: GalNAc alpha1-3Galbeta 1-4Gal, GalNAc
alpha1-3-Galbeta 1-4GlcNAc, 2'-modified-Gal alpha 1-3 Gal beta 1-4Gal or
2'-modified-Galalpha 1-3-Gal beta 1-4GlcNAc.

[0043] In another aspect, the invention features a composition comprising
a polypeptide fragment of an alpha 1,3 N-Acetylgalactosylaminotransferase
(alpha 3GalNaCT) that retains the ability to transfer a sugar with a
chemically reactive functional group from a sugar donor to a sugar
acceptor.

[0044] In one embodiment, the polypeptide fragment comprises a donor
substrate binding site, a hinge region and a DXD motif. In another
embodiment, the polypeptide fragment comprises one or more substitutions
in the donor substrate binding site. In another embodiment, the
polypeptide fragment comprises one or more substitutions in the hinge
region. In another embodiment, the polypeptide fragment comprises one or
more substitutions near the DXD motif.

[0045] In a related embodiment, the one or more substitutions in the
substrate binding site comprise an amino acid substitution at position
280, 281, or 282 corresponding to bovine alpha 1,3 galactosyltransferase
(alpha 3 Gal-T) (SEQ ID NO: 21). In another related embodiment, the one
or more substitutions in the substrate hinge region comprise an amino
acid substitution at position 191 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In one embodiment,
the one or more substitutions close to the DXD motif comprise an amino
acid substitution at position 228 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21). In another
embodiment, wherein a serine (S) is substituted for a histidine (H) at
amino acid position 280, a glycine (G) is substituted for an alanine (A)
at amino acid position 281, or a glycine (G) is substituted for an
alanine at amino acid position 282 of (SEQ ID NO 21). In another
embodiment, a serine (S) or an alanine (A) is substituted for a proline
(P) at amino acid position 191 corresponding to (SEQ ID NO: 21). In still
another embodiment, a glutamine (Q) is replaced with a a methionine (M)
at amino acid position 228 of (SEQ ID NO:21).

[0047] In one embodiment, the sugar acceptor is selected from the group
consisting of galactose beta 1,4 GlcNAc and galactose beta 1,4 glucose.
In another embodiment, the sugar with a chemically reactive functional
group is selected from the group consisting of UDP-GalNAc, UDP-galactose,
and UDP-galactose analogues. In another embodiment, the UDP-galactose
analogue comprises an azido group, a keto group or a thiol group. In
another embodiment, the keto group or the thiol group is substituted at
the C2 position of galactose. In another embodiment, one or more agents
are linked to a sugar moiety of the sugar donor. In a related embodiment,
the one or more agents is selected from the group consisting of: single
chain antibodies, bacterial toxins, growth factors, therapeutic agents,
targeting agents, contrast agents, chemical labels, radiolabels, and
fluorescent labels.

[0048] In another aspect, the invention features a composition comprising
a polypeptide fragment of an alpha 1,3 N-Acetylgalactosaminyltransferase
(alpha3GalNac-T) that retains that ability to transfer a sugar with a
chemically reactive functional group from a sugar donor to a sugar
acceptor and catalyzes the formation of an oligosaccharide.

[0049] In one embodiment, the oligosaccharide is a disaccharide or a
trisaccharide. In another embodiment, the trisaccharide is selected from
the group consisting of: GalNAc alpha1-3Galbeta 1-4Gal, GalNAc alpha
1-3-Galbeta 1-4GlcNAc, 2'-modified-Gal alpha 1-3 Gal beta 1-4Gal or
2'-modified-Galalpha 1-3-Gal beta 1-4GlcNAc.

[0051] In another aspect, the invention features an immunological
composition comprising a polypeptide fragment of an alpha 1,3
N-Acetylgalactosylaminotransferase (alpha 3GalNaCT) that retains the
ability to transfer a sugar with a chemically reactive functional group
from a sugar donor to a sugar acceptor.

[0052] In one embodiment, the polypeptide fragment comprises a donor
substrate-binding site, a hinge region and a DXD motif. In another
embodiment, the polypeptide fragment comprises one or more substitutions
in the donor substrate-binding site. In another embodiment, the
polypeptide fragment comprises one or more substitutions in the hinge
region. In another embodiment, the polypeptide fragment comprises one or
more substitutions near the DXD motif.

[0053] In a related embodiment, the one or more substitutions in the
substrate binding site comprise an amino acid substitution at position
280, 281, or 282 corresponding to bovine alpha 1,3 galactosyltransferase
(alpha 3 Gal-T) (SEQ ID NO: 21). In another related embodiment, the one
or more substitutions in the substrate hinge region comprise an amino
acid substitution at position 191 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In one embodiment,
the one or more substitutions close to the DXD motif comprise an amino
acid substitution at position 228 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21). In another
embodiment, wherein a serine (S) is substituted for a histidine (H) at
amino acid position 280, a glycine (G) is substituted for an alanine (A)
at amino acid position 281, or a glycine (G) is substituted for an
alanine at amino acid position 282 of (SEQ ID NO 21). In another
embodiment, a serine (S) or an alanine (A) is substituted for a proline
(P) at amino acid position 191 corresponding to (SEQ ID NO: 21). In still
another embodiment, a glutamine (Q) is replaced with a methionine (M) at
amino acid position 228 of (SEQ ID NO:21).

[0055] In one embodiment, the sugar acceptor is selected from the group
consisting of galactose beta 1,4 GlcNac and galactose beta 1,4 glucose.
In another embodiment, the sugar with a chemically reactive functional
group is selected from the group consisting of UDP-GalNAc, UDP-galactose,
and UDP-galactose analogues. In another embodiment, the UDP-galactose
analogue comprises an azido group, a keto group or a thiol group. In
another further embodiment, the azido group, the keto group or the thiol
group is substituted at the C2 position of galactose.

[0056] In another embodiment, one or more agents are linked to a sugar
moiety of the sugar donor. In another embodiment, the agent is selected
from single chain antibodies, monoclonal antibodies, polyclonal
antibodies, and chimeric antibodies.

[0057] In another aspect, the invention features an immunological
composition comprising a polypeptide fragment of an alpha 1,3
N-Acetylgalactosaminyltransferase (alpha3GalNac-T) that retains that
ability to transfer a sugar with a chemically reactive functional group
from a sugar donor to a sugar acceptor and catalyzes the formation of an
oligosaccharide, and wherein one or more antibodies are conjugated to the
chemically reactive functional group.

[0059] In another aspect the invention features a method of coupling an
agent to a carrier protein comprising incubating a reaction mixture
comprising A polypeptide fragment of an alpha 1,3
N-Acetylgalactosylaminotransferase (alpha 3GalNaCT) that retains the
ability to transfer a sugar with a chemically reactive functional group
from a sugar donor to a sugar acceptor, wherein the sugar donor is
coupled to an agent and the sugar acceptor is a carrier protein.

[0060] In another embodiment the polypeptide fragment comprises a donor
substrate binding site, a hinge region and a DXD motif. In another
embodiment, the polypeptide fragment comprises one or more substitutions
in the donor substrate-binding site. In another embodiment, the
polypeptide fragment comprises one or more substitutions in the hinge
region. In another embodiment, the polypeptide fragment comprises one or
more substitutions near the DXD motif.

[0061] In a related embodiment, the one or more substitutions in the
substrate binding site comprise an amino acid substitution at position
280, 281, or 282 corresponding to bovine alpha 1,3 galactosyltransferase
(alpha 3 Gal-T) (SEQ ID NO: 21). In another related embodiment, the one
or more substitutions in the substrate hinge region comprise an amino
acid substitution at position 191 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In one embodiment,
the one or more substitutions close to the DXD motif comprise an amino
acid substitution at position 228 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21). In another
embodiment, wherein a serine (S) is substituted for a histidine (H) at
amino acid position 280, a glycine (G) is substituted for an alanine (A)
at amino acid position 281, or a glycine (G) is substituted for an
alanine at amino acid position 282 of (SEQ ID NO 21). In another
embodiment, a serine (S) or an alanine (A) is substituted for a proline
(P) at amino acid position 191 corresponding to (SEQ ID NO: 21). In still
another embodiment, a glutamine (Q) is replaced with a a methionine (M)
at amino acid position 228 of (SEQ ID NO:21).

[0062] In another embodiment, the sugar acceptor is selected from the
group consisting of galactose beta 1,4 GlcNac and galactose beta 1,4
glucose.

[0063] In another embodiment, the sugar with a chemically reactive
functional group is selected from the group consisting of UDP-GalNAc,
UDP-galactose, and UDP-galactose analogues. In a related embodiment, the
UDP-galactose analogue comprises an azido group, a keto group or a thiol
group. In another related embodiment, the azido group, the keto group or
the thiol group is substituted at the C2 position of galactose.

[0064] In another embodiment, one or more agents are linked to a sugar
moiety of the sugar donor.

[0065] In another embodiment, the one or more agents is selected from the
group consisting of: single chain antibodies, bacterial toxins, growth
factors, therapeutic agents, targeting agents, contrast agents,
paramagnetic contrast agents, chemical labels, radiolabels, and
fluorescent labels.

[0066] In one embodiment, the oligosaccharide is a disaccharide or a
trisaccharide. In another embodiment, the trisaccharide is selected from
the group consisting of: GalNAc alpha1-3Galbeta 1-4Gal, GalNAc
alpha1-3-Galbeta 1-4GlcNAc, 2'-modified-Gal alpha 1-3 Gal beta 1-4Gal or
2'-modified-Galalpha 1-3-Gal beta 1-4GlcNAc.

[0070] In a related embodiment, the method of any of the above-mentioned
aspects is used in imaging.

[0071] In another related embodiment, the agent is a paramagnetic imaging
agent used in magnetic resonance imaging.

[0072] In another aspect, the invention features a method for the
diagnosis or treatment of a subject suffering from a disease or disorder
comprising administering to the subject an effective amount of an
isolated glycoprotein synthesized by the method according to any one the
above-mentioned aspects, wherein one or more agents are linked to the
sugar donor, and thereby diagnosing or treating a subject suffering from
a disease or disorder.

[0073] In one embodiment, the disease or disorder is selected from the
group consisting of: proliferative diseases, cardiovascular diseases,
inflammatory diseases, cancer, diseases of ageing, and metabolic diseases
or disorders.

[0075] In another aspect, the invention features a method for imaging a
target cell or tissue comprising administering to a subject an
oligosaccharide synthesized by the method according to any one of the
above-mentioned aspects, and wherein one or more imaging agents are
linked to the sugar donor, thereby imaging a target cell or tissue.

[0076] In still another aspect, the invention features a method for
synthesizing a detectable Gal beta 1-4GlcNAc epitope comprising
synthesizing an oligosaccharide according to the method of any one of the
above-mentioned aspects, wherein the sugar donor comprises a 2' modified
Gal residue and wherein or more detection agents are linked to the 2'
modified Gal residue and thereby synthesizing a detectable Gal beta
1-4GlcNAc epitope.

[0077] In one embodiment, the detectable Gal beta 1-4GlcNAc epitope is
administered to a subject.

[0078] In another embodiment, the detectable Gal beta 1-4GlcNAc epitope is
administered to a subject to diagnose a disease or disorder.

[0079] In another embodiment, the disease or disorder selected from the
group consisting of: proliferative diseases, cardiovascular diseases,
inflammatory diseases, cancer, diseases of ageing, and metabolic diseases
or disorders.

[0080] In another aspect the invention features a method for inducing an
immune response in a subject comprising administering to the subject an
immunological composition according to any one of the above-mentioned
aspects.

[0081] In another aspect, the invention provides a kit comprising
packaging material, and an polypeptide fragment from an alpha 1,3
N-Acetylgalactosaminyltransferase (alpha3GalNac-T) according to any one
of the above-mentioned aspects.

[0082] In one embodiment, the kit comprises a sugar donor.

[0083] In another embodiment, the donor is selected from the group
consisting of UDP-galactose, UDP-GalNAc or UDP-GalNAc analogue. In a
related embodiment, an agent is linked to the sugar donor. In another
related embodiment, the agent is selected from the group consisting of:
single chain antibodies, bacterial toxins, growth factors, therapeutic
agents, contrast agents, targeting agents, chemical labels, a
radiolabels, and fluorescent labels.

BRIEF DESCRIPTION OF THE DRAWINGS

[0084]FIG. 1 is a schematic showing the structure--based design of
al-3-N-acetylgalactosaminyltransferase from al-3-galactosyltransferase.

[0085]FIG. 2 is a diagram illustrating the structure-based design of a1-3
N-acetylgalactosaminyltransferase (a3GalNAc-T) from
a1-3galactosyltransferase (a3Gal-T). The boxed region shows a
magnification of the sugar donor binding site and the hinge region where
the substitutions occur.

[0088]FIG. 5 is a table showing the effect of substitutions in the donor
substrate binding site, hinge region and near DXD motif on Gal activity,
GalNAc activity and GalKeto activity.

DETAILED DESCRIPTION OF THE INVENTION

[0089] The invention generally features compositions and methods based on
the structure-based design of alpha 1-3 N-acetylgalactosaminyltransferase
(alpha 3 GalNAc-T) enzymes from alpha 1-3galactosyltransferase (a3Gal-T)
that can transfer 2'-modified galactose from the corresponding
UDP-derivatives due to substitutions that broaden the alpha 3Gal-T donor
specificity and make the enzyme a3 GalNAc-T.

DEFINITIONS

[0090] Unless defined otherwise, all technical and scientific terms used
herein have the meaning commonly understood by a person skilled in the
art to which this invention belongs. The following references provide one
of skill with a general definition of many of the terms used in this
invention: Singleton et al., Dictionary of Microbiology and Molecular
Biology (2nd ed. 1994); The Cambridge Dictionary of Science and
Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.
Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The
Harper Collins Dictionary of Biology (1991). As used herein, the
following terms have the meanings ascribed to them below, unless
specified otherwise.

[0091] As used in the specification and claims, the singular form "a",
"an" and "the" include plural references unless the context clearly
dictates otherwise. For example, the term "a cell" includes a plurality
of cells, including mixtures thereof. The term "a nucleic acid molecule"
includes a plurality of nucleic acid molecules.

[0092] As used herein, the term "comprising" is intended to mean that the
compositions and methods include the recited elements, but do not exclude
other elements. "Consisting essentially of", when used to define
compositions and methods, shall mean excluding other elements of any
essential significance to the combination. Thus, a composition consisting
essentially of the elements as defined herein would not exclude trace
contaminants from the isolation and purification method and
pharmaceutically acceptable carriers, such as phosphate buffered saline,
preservatives, and the like. "Consisting of" shall mean excluding more
than trace elements of other ingredients and substantial method steps for
administering the compositions of this invention. Embodiments defined by
each of these transition terms are within the scope of this invention.

[0093] The term "acceptor" is meant to refer to a molecule or structure
onto which a donor is actively linked through action of a catalytic
domain of a galactosyltransferase, or mutant thereof. Examples of
acceptors include, but are not limited to, carbohydrates, glycoproteins,
glycolipids. The acceptor polypeptide can comprise, in preferred
embodiments Galactose residues, free or attached to a peptide or
glycopeptide.

[0094] The term "agent" or "bioactive agent" is meant to refer to any
chemical or biological material or compound that is suitable for delivery
that induces a desired effect in or on an organism, such as a biological
or pharmacological effect, which may include, but is not limited to a
prophylactic effect, alleviating a condition caused by a disease or a
disorder, reducing or eliminating a disease or disorder. An agent or a
bioactive agent refers to substances that are capable of exerting a
biological effect in vitro and/or in vivo. Examples include diagnostic
agents, pharmaceuticals, drugs, synthetic organic molecules, proteins,
peptides, vitamins, steroids, genetic material including nucleotides,
nucleosides, polynucleotides, RNAs, siRNAs, shRNAs, anti-sense DNA or
RNA.

[0095] The term "antibody" as used herein refers to both polyclonal and
monoclonal antibody. Te term can also refer to single chain antibodies.
The term encompasses not only intact immunoglobulin molecules, but
fragments and genetically engineered derivatives of immunoglobulin
molecules as may be prepared by techniques known in the art, and which
retains the binding specificity of the antigen binding site.

[0097] The term "donor" refers to a molecule that is actively linked to an
acceptor molecule through the action of a catalytic domain of a
galactosyltransferase, or mutant thereof. A donor molecule can include a
sugar, or a sugar derivative. Examples of donors include, but are not
limited to, UDP-GalNAc, UDP-galactose or UDP-galNAc analogues,
UDP-galactose analogues. Donors include sugar derivatives that include
agents, biological agents, or active groups. Accordingly,
oligosaccharides may be prepared according to the methods of the
invention that include a sugar derivative having any desired
characteristic.

[0098] The term "DXD motif" is meant to refer to a glycosyltransferase
sugar-binding region containing DXD motif. In preferred embodiments, the
DXD motif is a short conserved motif found in many families of
glycosyltransferases, which add a range of different sugars to other
sugars, phosphates and proteins. In other certain embodiments,
DXD-containing glycosyltransferases all use nucleoside diphosphate sugars
as donors and require divalent cations, usually manganese. Preferred DXD
motifs are represented by the NCBI conserved domain database designation
pfam04488.8.

[0099] The term "effective amount" is meant to refer to a sufficient
amount that is capable of providing the desired local or systemic effect.

[0100] The term "expression cassette" as used herein refers to a DNA
sequence capable of directing expression of a particular nucleotide
sequence in an appropriate host cell, comprising a promoter operably
linked to the nucleotide sequence of interest that is operably linked to
termination signals. It also typically comprises sequences required for
proper translation of the nucleotide sequence. The expression cassette
may be one that is naturally occurring but has been obtained in a
recombinant form useful for heterologous expression. The expression of
the nucleotide sequence in the expression cassette may be under the
control of a constitutive promoter or of an inducible promoter that
initiates transcription only when the host cell is exposed to some
particular external stimulus. In the case of a multicellular organism,
the promoter can also be specific to a particular tissue or organ or
stage of development.

[0101] The term "alpha1-3 N-acetylgalactosaminyltransferase (a3 GalNAc-T)"
as used herein refers to enzymes substantially homologous to, and having
substantially the same biological activity as, the enzyme coded for by
the nucleotide sequence depicted in any one of SEQ ID NOs: 1, 3, 5, 7, 9,
11, 15, 17, 19 and the amino acid sequence depicted in SEQ ID NOs: 2, 4,
6, 8, 10, 12, 14, 16, 18, 20. This definition is intended to encompass
natural allelic variations in the a3 GalNAc-T sequence, and all
references to a3 GalNAc-T, and nucleotide and amino acid sequences
thereof are intended to encompass such allelic variations, both naturally
occurring and man-made. The production of proteins such as the enzyme a3
GalNAc-T from cloned genes by genetic engineering is well known.

[0102] The a3 GalNAc-T enzyme may be synthesized in host cells transformed
with vectors containing DNA encoding the a3 GalNAc-T enzyme. A vector is
a replicable DNA construct. Vectors are used herein either to amplify DNA
encoding the a3 GalNAc-T enzyme and/or to express DNA which encodes the
a3 GalNAc-T enzyme. An expression vector is a replicable DNA construct in
which a DNA sequence encoding the a3 GalNAc-T enzyme is operably linked
to suitable control sequences capable of effecting the expression of the
a3 GalNAc-T enzyme in a suitable host. The need for such control
sequences will vary depending upon the host selected and the
transformation method chosen. Generally, control sequences include a
transcriptional promoter, an optional operator sequence to control
transcription, a sequence encoding suitable mRNA ribosomal binding sites,
and sequences which control the termination of transcription and
translation. Amplification vectors do not require expression control
domains. All that is needed is the ability to replicate in a host,
usually conferred by an origin of replication, and a selection gene to
facilitate recognition of transformants.

[0103] The term "homologous" is intended to include a first amino acid or
nucleotide sequence which contains a sufficient or minimum number of
identical or equivalent amino acid residues or nucleotides, e.g., an
amino acid residue which has a similar side chain, to a second amino acid
or nucleotide sequence such that the first and second amino acid or
nucleotide sequences share common structural domains and/or a common
functional activity.

[0104] The terms "oligosaccharide" and "polysaccharide" are used
interchangeably herein. These terms refer to saccharide chains having two
or more linked sugars. Oligosaccharides and polysaccharides may be
homopolymers and heteropolymers having a random sugar sequence or a
preselected sugar sequence. Additionally, oligosaccharides and
polysaccharides may contain sugars that are normally found in nature,
derivatives of sugars, and mixed polymers thereof. "saccharide" refers to
any of a series of compounds of carbon, hydrogen, and oxygen in which the
atoms of the latter two elements are in the ratio of 2:1, especially
those containing the groupC6H1o05, including fructose, glucose, sucrose,
lactose, maltose, galactose and arabinose.

[0105] The term "immunogenic" compound or composition as used herein
refers to a compound or composition that is capable of stimulating
production of a specific immunological response when administered to a
suitable host, usually a mammal.

[0106] The term "nucleic acid" is intended to include nucleic acid
molecules, e.g., polynucleotides which include an open reading frame
encoding a polypeptide, and can further include non-coding regulatory
sequences, and introns. In addition, the terms are intended to include
one or more genes that map to a functional locus. In addition, the terms
are intended to include a specific gene for a selected purpose. The gene
can be endogenous to the host cell or can be recombinantly introduced
into the host cell, e.g., as a plasmid maintained episomally or a plasmid
(or fragment thereof) that is stably integrated into the genome. In one
embodiment, the gene of polynucleotide segment is involved sugar
transfer. A mutant nucleic acid molecule or is intended to include a
nucleic acid molecule or gene having a nucleotide sequence which includes
at least one alteration (e.g., substitution, insertion, deletion) such
that the polypeptide or polypeptide that can be encoded by said mutant
exhibits an activity that differs from the polypeptide or polypeptide
encoded by the wild-type nucleic acid molecule or gene.

[0107] The terms "polypeptides" or "isolated polypeptide" and "proteins"
are used interchangeably herein. Polypeptides and proteins can be
expressed in vivo through use of prokaryotic or eukaryotic expression
systems. Many such expressions systems are known in the art and are
commercially available. (Clontech, Palo Alto, Calif.; Stratagene, La
Jolla, Calif.). Examples of such systems include, but are not limited to,
the T7-expression system in prokaryotes and the bacculovirus expression
system in eukaryotes. Polypeptides can also be synthesized in vitro,
e.g., by the solid phase peptide synthetic method or by in vitro
transcription/translation systems. Such methods are described, for
example, in U.S. Pat. Nos. 5,595,887; 5,116,750; 5,168,049 and 5,053,133;
Olson et al., Peptides, 9, 301, 307 (1988). The solid phase peptide
synthetic method is an established and widely used method, which is
described in the following references: Stewart et al., Solid Phase
Peptide Synthesis, W.H. Freeman Co., San Francisco (1969); Merrifield, J.
Am. Chem. Soc., 85 2149 (1963); Meienhofer in "Hormonal Proteins and
Peptides," ed.; C. H. Li, Vol. 2 (Academic Press, 1973), pp. 48-267;
Bavaay and Merrifield, "The Peptides," eds. E. Gross and F. Meienhofer,
Vol. 2 (Academic Press, 1980) pp. 3-285; and Clark-Lewis et al., Meth.
Enzymol., 287, 233 (1997). These polypeptides can be further purified by
fractionation on immunoaffinity or ion-exchange columns; ethanol
precipitation; reverse phase HPLC; chromatography on silica or on an
anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium
sulfate precipitation; gel filtration using, for example, Sephadex G-75;
or ligand affinity chromatography. The term an "isolated polypeptide"
(e.g., an isolated or purified biosynthetic enzyme) is substantially free
of cellular material or other contaminating polypeptides from the
microorganism from which the polypeptide is derived, or substantially
free from chemical precursors or other chemicals when chemically
synthesized

[0108] The polypeptides of the invention include polypeptides having amino
acid exchanges, i.e., variant polypeptides, so long as the polypeptide
variant is biologically active. The variant polypeptides include the
exchange of at least one amino acid residue in the polypeptide for
another amino acid residue, including exchanges that utilize the D rather
than L form, as well as other well known amino acid analogs, e.g.,
N-alkyl amino acids, lactic acid, and the like. These analogs include
phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline,
gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid,
statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,
penicillamine, ornithine, citruline, N-methyl-alanine,
para-benzoyl-phenylalanine, phenylglycine, propargylglycine, sarcosine,
N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,
and other similar amino acids and imino acids and tert-butylglycine.

[0109] Conservative amino acid exchanges are preferred and include, for
example; aspartic-glutamic as acidic amino acids;
lysine/arginine/histidine as basic amino acids; leucine/isoleucine,
methionine/valine, alanine/valine as hydrophobic amino acids;
serine/glycine/alanine/threonine as hydrophilic amino acids. Conservative
amino acid exchange also includes groupings based on side chains. Members
in each group can be exchanged with another. For example, a group of
amino acids having aliphatic side chains is glycine, alanine, valine,
leucine, and isoleucine. These may be exchanged with one another. A group
of amino acids having aliphatic-hydroxyl side chains is serine and
threonine. A group of amino acids having amide-containing side chains is
asparagine and glutamine. A group of amino acids having aromatic side
chains is phenylalanine, tyrosine, and tryptophan. A group of amino acids
having basic side chains is lysine, arginine, and histidine. A group of
amino acids having sulfur-containing side chains is cysteine and
methionine. For example, replacement of a leucine with an isoleucine or
valine, an aspartate with a glutamate, a threonine with a serine, or a
similar replacement of an amino acid with a structurally related amino
acid may be accomplished to produce a variant polypeptide of the
invention.

[0110] The term "subject" as used herein refers to any animal, including
mammals, preferably humans, to which the present invention may be
applied.

[0111] The term "cancer" or "tumor" refers to an aggregate of abnormal
cells and/or tissue which may be associated with diseased states that are
characterized by uncontrolled cell proliferation. The disease states may
involve a variety of cell types, including, for example, endothelial,
epithelial and myocardial cells. Included among the disease states are
neoplasms, cancer, leukemia and restenosis injuries.

Alpha 1,3 N-Acetylgalactosaminyltransferase (a3 GalNAc-T)

[0112] Specific glycosyltransferases synthesize oligosaccharides by the
sequential transfer of the monosaccharide moiety of an activated sugar
donor to an acceptor molecule. Members of the glycosyltransferase
superfamily, which are often named after the sugar moiety that they
transfer, are divided into subfamilies on the basis of linkage that is
generated between the donor and acceptor. Transfer of the sugar residue
occurs with either the retention (by retaining glycosyltransferases) or
the inversion (by inverting glycosyltransferases) of the configuration at
the anomeric C1 atom. Glycosyltransferases show great structural
similarity. They are all globular proteins with two types of fold, termed
GT-A and GT-B, which each have an N-terminal and a C-terminal domain. The
enzymes of the GT-A fold have two dissimilar domains. The N-terminal
domain, which recognizes the sugar-nucleotide donor, comprises several
b-strands that are each flanked by a-helices as in a Rossmann-like fold,
whereas the C-terminal domain, which contains the acceptor-binding site,
consists largely of mixed b-sheets. By contrast, enzymes with the GT-B
fold contain two similar Rossmann-like folds, with the N-terminal domain
providing the acceptor-binding site and the C-terminal domain providing
the donor-binding site. In both types of enzyme, the two domains are
connected by a linker region and the active site is located between the
two domains. A metal-binding site is also located in the cleft in enzymes
of both the GT-B and GT-A fold (Qasba et al. 2005).

[0113] Alpha (1,3)-galactosyltransferase I (a3 Gal-7)

[0114] The alpha (1,3)-galactosyltransferase I (a3 Gal-T) enzyme mediates
the formation of gal-alpha-gal moieties. A3 Gal-T uses UDP-galactose as a
source of galactose, which it transfers to an acceptor oligosaccharide,
usually Gal beta (1,4)GlcNAc (N-acetyl lactosamine). As used herein the
term "alpha (1,3)galactosyltransferase" and the abbreviation "alpha
1,3GT" refer to the enzyme, present in non-primate mammals, that
catalyzes the formation of the Galα (1,3)Gal determinant by
attaching Gal in the α (1,3) position to the Galβ (1,4)GlcNAc
acceptorα1,3GT has the Enzyme Commission designation EC 2.4.1.124.

[0116] The term Gal alpha (1,3)Gal refers to an oligosaccharide
determinant present on endothelial cells and other cells of most
non-primate mammals, for which humans have a naturally occurring
antibody. Except for Old World monkeys, apes and humans, most mammals
carry glycoproteins on their cell surfaces that contain galactose alpha
1,3-galactose (Galili et al., J. Biol. Chem. 263: 17755-17762, 1988).
Humans, apes and Old World monkeys have a naturally occurring anti-alpha
gal antibody that is produced in high quantity (Cooper et al., Lancet
342:682-683, 1993). It binds specifically to glycoproteins and
glycolipids bearing galactose alpha-1,3 galactose. In contrast,
glycoproteins that contain galactose alpha 1,3-galactose are found in
large amounts on cells of other mammals, such as pigs. This differential
distribution of the "alpha-1,3 GT epitope" and anti-Gal antibodies (i.e.,
antibodies binding to glycoproteins and glycolipids bearing galactose
alpha-1,3 galactose) in mammals is the result of an evolutionary process
which selected for species with inactivated (i.e. mutated)
alpha-1,3-galactosyltransferase in ancestral Old World primates and
humans. Thus, humans are "natural knockouts" of alpha-1,3GT. A direct
outcome of this event is the rejection of xenografts, such as the
rejection of pig organs transplanted into humans initially via HAR.

[0117] Alpha 1,3 N-Acetylgalactosaminyltransferase (a3 GalNAc-T)

[0118] The present invention features the structure-based design of alpha
1-3 N-Acetylgalactosaminyltransferase (a3 GalNAc-T) from a3Gal-T that can
transfer 2'-modified galactose from the corresponding UDP-derivatives.
The genetically engineered a3Gal-T to a3GalNAc-T, which can transfer
2'N-acetygalactose (GalNAc) or 2'-modified galactose from the
corresponding UDP-derivatives, is very useful for the synthesis of a
trisaccharide GalNAcαl-3Galβ 1-4Glc or GalNAcαl-3-Gal
β1-4GlcNAc or 2'-modified-Galα1-3Gal β1-4Glc or
2'-modified-Galα1-3-Gal β1-4GlcNAc in an oligosaccharide chain
that is otherwise difficult to be synthesized by chemical methods.

[0119] The Sugar Binding Pocket

[0120] It has been discovered that mutation of alpha 1,3
N-Acetylgalactosaminyltransferase (a3 GalNAc-T) can broaden the donor
specificity of the enzyme. More specifically, it has been determined
that, in certain embodiments, mutation in residues in the
sugar-nucleotide binding pocket of the enzyme can broaden the donor
specificity of the enzyme. In particular, substitution of amino acid
residues located in the in the sugar-nucleotide binding pocket provide
greater flexibility and decreased steric hindrance that allow a broader
range of donor (or substrate) binding, for example UDP-GalNAc, UDP-galNAc
analogues, UDP-galactose, or UDP-galactose analogues, while still
preserving interaction with amino acid residues active during catalytic
bond formation between the donor and the acceptor.

[0121] A three-residue motif, Asp-X-Asp (DXD) or Glu-X-Asp (EXD), or its
equivalent generally participates in metal ion binding in enzymes of the
GT-A fold. Enzymes of the GT-B fold such as the microbial
glycosyltransferases MurG (Hu, Y. et al. (2003)) and GtfB (Mulichack et
al. 2001), and BGT (Morera et al. 1999), do not have a DXD motif or its
equivalent, even though some, BGT for example, require a metal ion for
activity. In glycosyltransferases that require Mn2C ion as cofactor, the
metal ion is bound in an octahedral coordination (Qasba et al. 2005). It
interacts with one or both acidic residues of the DXD or EXD motif and
with two oxygen atoms from the a-phosphate and b-phosphate

of UDP. To satisfy the octahedral geometry, the three remaining metal ion
links are made either to water molecules or to water in combination with
other residues of the protein. In several glycosyltransferases only the
first (Lobsanov, Y. D. et al. (2004)) or the second (Gastinel et al.
1999; Ramakrishnan et al. 2001; Ramakrishnan 2002; Unligil 2000) acidic
residue of the motif coordinates directly with the metal ion. For
example, in some enzymes, the first acidic residue of the motif either
interacts directly with the sugar donor or the ribose moiety or interacts
via the water molecules coordinated to the Mn2C ion. In blood group A and
B and alpha 3GT transferases, by contrast, both aspartic acid residues of
the DXD motif directly coordinate the metal ion.

[0122] The crystal structures of several glycosyltransferases of either
the GT-A or GT-B fold show that at least one flexible loop region has a
crucial role in the catalytic mechanism of the enzyme (Qasba et al.
2005). Although the exact location of this loop differs among the
transferases, it is invariably located in the vicinity of the
sugamucleotide-binding site. Owing to the flexibility of this region, the
loop structure cannot be traced in the apo form of the enzyme, which
lacks bound substrate. In the sugar-nucleotide-bound structures, the loop
either is in a closed conformation covering the bound donor substrate or
is found disordered in the vicinity of the sugamucleotide-binding site.
In a3GT, the C-terminal 11-residue flexible loop changes its conformation
when the sugamucleotide donor is bound (Boix et al., 2001).

[0123] Without being bound by any theory, examples of catalytic residues
thought to be important for binding include Pro 191, Gln228, His280,
A1a28 1, and A1a282 of bovine a3 GalT. Accordingly, the invention
provides alpha 1,3 N-Acetylgalactosaminyltransferase (a3 GalNAc-T)
enzymes having amino acid substitutions, insertions, and deletions that
provide greater flexibility and decreased steric hindrance in the sugar
nucleotide binding pocket to allow the mutated alpha 1,3
N-Acetylgalactosaminyltransferase (a3 GalNAc-T) to catalyze chemical
bonding of the donor to an acceptor, such as N-acetylglucosamine
(GlcNAc), galactose (Gal) and xylose residues of glycoproteins,
glycolipids or proteoglycan (glycoconjugates).

[0124] Polypeptide Fragments

[0125] The invention features, in certain embodiments, polypeptide
fragments from alpha 1,3 N-Acetylgalactosaminyltransferases
(alpha3GalNac-T) transfer sugars with a chemically reactive functional
group from a sugar donor to a sugar acceptor. The a3 GalNAc transferases
described herein comprise substitutions in a3 Gal-T, in certain preferred
examples, bovine a 3GalT. The substitutions have the effect of broadening
the donor specificity of the transferase and make the enzyme an alpha 3
GalNAc-T.

[0126] In certain examples, the invention provides a polypeptide fragment
of an alpha 1,3 N-Acetylgalactosylaminotransferase (alpha 3GalNaCT) that
retains the ability to transfer a sugar with a chemically reactive
functional group from a sugar donor to a sugar acceptor. The polypeptide
fragments, in certain preferred examples, comprise a donor
substrate-binding site, a hinge region and a DXD motif. Thr polypeptide
fragment can comprise one or more substitutions in the donor
substrate-binding site.

[0127] A number of substitutions of the polypeptide fragments are
envisioned by the instant invention. The substitutions have the effect of
broadening the donor specificity of the enzyme. In certain examples, the
polypeptide fragment comprises one or more substitutions in the hinge
region. In other examples, polypeptide fragment comprise one or more
substitutions near the DXD motif. the one or more substitutions in the
substrate binding site comprise an amino acid substitution at position
280, 281, or 282 corresponding to bovine alpha 1,3 galactosyltransferase
(alpha 3 Gal-T) (SEQ ID NO: 21). In another related embodiment, the one
or more substitutions in the substrate hinge region comprise an amino
acid substitution at position 191 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In one embodiment,
the one or more substitutions close to the DXD motif comprise an amino
acid substitution at position 228 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21). In another
embodiment, wherein a serine (S) is substituted for a histidine (H) at
amino acid position 280, a glycine (G) is substituted for an alanine (A)
at amino acid position 281, or a glycine (G) is substituted for an
alanine at amino acid position 282 of (SEQ ID NO 21). In another
embodiment, a serine (S) or an alanine (A) is substituted for a proline
(P) at amino acid position 191 corresponding to (SEQ ID NO: 21). In still
another embodiment, a glutamine (Q) is replaced with a a methionine (M)
at amino acid position 228 of (SEQ ID NO:21).

[0140] An isolated gene includes a gene which is essentially free of
sequences which naturally flank the gene in the chromosomal DNA of the
organism from which the gene is derived (i.e., is free of adjacent coding
sequences which encode a second or distinct polypeptide or RNA molecule,
adjacent structural sequences or the like) and optionally includes 5' and
3' regulatory sequences, for example promoter sequences and/or terminator
sequences.

[0144] As described herein, the present invention provides isolated
nucleic acid segments that encode polypeptide fragments of alpha1-3
N-Acetylgalactosaminyltransferase (alpha 3 GalNAc-T). In certain
embodiments, for example, the substitutions are in bovine a3Gal-T.

[0145] In certain examples, the one or more substitutions in the substrate
binding site comprise an amino acid substitution at position 280, 281, or
282 corresponding to bovine alpha 1,3 galactosyltransferase (alpha 3
Gal-T) (SEQ ID NO: 21). In another related embodiment, the one or more
substitutions in the substrate hinge region comprise an amino acid
substitution at position 191 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In one embodiment,
the one or more substitutions close to the DXD motif comprise an amino
acid substitution at position 228 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21). In another
embodiment, wherein a serine (S) is substituted for a histidine (H) at
amino acid position 280, a glycine (G) is substituted for an alanine (A)
at amino acid position 281, or a glycine (G) is substituted for an
alanine at amino acid position 282 of (SEQ ID NO 21). In another
embodiment, a serine (S) or an alanine (A) is substituted for a proline
(P) at amino acid position 191 corresponding to (SEQ ID NO: 21). In still
another embodiment, a glutamine (Q) is replaced with a methionine (M) at
amino acid position 228 of (SEQ ID NO: 21).

[0147] The nucleic acid segments can be inserted into numerous types of
vectors. A vector may include, but is not limited to, any plasmid,
phagemid, F-factor, virus, cosmid, or phage in double or single stranded
linear or circular form, which may or may not be self-transmissible or
mobilizable. The vector can also transform a prokaryotic or eukaryotic
host either by integration into the cellular genome or exist
extrachromosomally (e.g. autonomous replicating plasmid with an origin of
replication).

[0148] Preferably the nucleic acid segment in the vector is under the
control of, and operably linked to, an appropriate promoter or other
regulatory elements for transcription in vitro or in a host cell such as
a eukaryotic cell or microbe, e.g. bacteria. The vector may be a
bi-functional expression vector which functions in multiple hosts. In the
case of genomic DNA, this may contain its own promoter or other
regulatory elements and in the case of cDNA this may be under the control
of a promoter or other regulatory sequences for expression in a host
cell.

[0149] Specifically included are shuttle vectors by which is meant a DNA
vehicle capable, naturally or by design, of replication in two different
host organisms, which may be selected from bacteria and eukaryotic cells
(e.g. mammalian, yeast or fungal).

[0150] The vector may also be a cloning vector which typically contains
one or a small number of restriction endonuclease recognition sites at
which nucleic acid segments can be inserted in a determinable fashion.
Such insertion can occur without loss of essential biological function of
the cloning vector. A cloning vector may also contain a marker gene that
is suitable for use in the identification and selection of cells
transformed with the cloning vector. Examples of marker genes are
tetracycline resistance, hygromycin resistance or ampicillin resistance.
Many cloning vectors are commercially available (Stratagene, New England
Biolabs, Clonetech).

[0151] The nucleic acid segments of the invention may also be inserted
into an expression vector. Typically an expression vector contains (1)
prokaryotic DNA elements coding for a bacterial replication origin and an
antibiotic resistance gene to provide for the amplification and selection
of the expression vector in a bacterial host; (2) regulatory elements
that control initiation of transcription such as a promoter; and (3) DNA
elements that control the processing of transcripts such as introns,
transcription termination/polyadenylation sequence.

[0152] Methods to introduce a nucleic acid segment into a vector are well
known in the art (Sambrook et al., 1989). Briefly, a vector into which
the nucleic acid segment is to be inserted is treated with one or more
restriction enzymes (restriction endonuclease) to produce a linearized
vector having a blunt end, a "sticky" end with a 5' or a 3' overhang, or
any combination of the above. The vector may also be treated with a
restriction enzyme and subsequently treated with another modifying
enzyme, such as a polymerase, an exonuclease, a phosphatase or a kinase,
to create a linearized vector that has characteristics useful for
ligation of a nucleic acid segment into the vector. The nucleic acid
segment that is to be inserted into the vector is treated with one or
more restriction enzymes to create a linearized segment having a blunt
end, a "sticky" end with a 5' or a 3' overhang, or any combination of the
above. The nucleic acid segment may also be treated with a restriction
enzyme and subsequently treated with another DNA modifying enzyme. Such
DNA modifying enzymes include, but are not limited to, polymerase,
exonuclease, phosphatase or a kinase, to create a polynucleic acid
segment that has characteristics useful for ligation of a nucleic acid
segment into the vector.

[0153] The treated vector and nucleic acid segment are then ligated
together to form a construct containing a nucleic acid segment according
to methods known in the art (Sambrook, 2002). Briefly, the treated
nucleic acid fragment and the treated vector are combined in the presence
of a suitable buffer and ligase. The mixture is then incubated under
appropriate conditions to allow the ligase to ligate the nucleic acid
fragment into the vector. It is preferred that the nucleic acid fragment
and the vector each have complimentary "sticky" ends to increase ligation
efficiency, as opposed to blunt-end ligation. It is more preferred that
the vector and nucleic acid fragment are each treated with two different
restriction enzymes to produce two different complimentary "sticky" ends.
This allows for directional ligation of the nucleic acid fragment into
the vector, increases ligation efficiency and avoids ligation of the ends
of the vector to reform the vector without the inserted nucleic acid
fragment.

[0154] Suitable prokaryotic vectors include but are not limited to pBR322,
pMB9, pUC, lambda bacteriophage, m13 bacteriophage, and Bluescript®.
Suitable eukaryotic vectors include but are not limited to PMSG,
pAV009/A+, PMTO10/A+, pMAM neo-5, bacculovirus, pDSVE, YIPS, YRP17, YEP.
It will be clear to one of ordinary skill in the art which vector or
promoter system should be used depending on which cell type is used for a
host cell.

[0155] The invention also provides expression cassettes which contain a
control sequence capable of directing expression of a particular nucleic
acid segment of the invention either in vitro or in a host cell. The
expression cassette is an isolatable unit such that the expression
cassette may be in linear form and functional in in vitro transcription
and translation assays. The materials and procedures to conduct these
assays are commercially available from Promega Corp. (Madison, Wis.). For
example, an in vitro transcript may be produced by placing a nucleic acid
segment under the control of a T7 promoter and then using T7 RNA
polymerase to produce an in vitro transcript. This transcript may then be
translated in vitro through use of a rabbit reticulocyte lysate.
Alternatively, the expression cassette can be incorporated into a vector
allowing for replication and amplification of the expression cassette
within a host cell or also in vitro transcription and translation of a
nucleic acid segment.

[0156] Such an expression cassette may contain one or a plurality of
restriction sites allowing for placement of the nucleic acid segment
under the regulation of a regulatory sequence. The expression cassette
can also contain a termination signal operably linked to the nucleic acid
segment as well as regulatory sequences required for proper translation
of the nucleic acid segment. Expression of the nucleic acid segment in
the expression cassette may be under the control of a constitutive
promoter or an inducible promoter, which initiates transcription only
when the host cell is exposed to some particular external stimulus.

[0158] The regulatory sequence can be a nucleic acid sequence located
upstream (5' non-coding sequences), within, or downstream (3' non-coding
sequences) of a coding sequence, and which influences the transcription,
RNA processing or stability, or translation of the associated coding
sequence. Regulatory sequences can include, but are not limited to,
enhancers, promoter and repressor binding sites, translation leader
sequences, introns, and polyadenylation signal sequences. They may
include natural and synthetic sequences as well as sequences that may be
a combination of synthetic and natural sequences. While regulatory
sequences are not limited to promoters, some useful regulatory sequences
include constitutive promoters, inducible promoters, regulated promoters,
tissue-specific promoters, viral promoters and synthetic promoters.

[0159] A promoter is a nucleotide sequence that controls expression of the
coding sequence by providing the recognition for RNA polymerase and other
factors required for proper transcription. A promoter includes a minimal
promoter, consisting only of all basal elements needed for transcription
initiation, such as a TATA-box and/or initiator that is a short DNA
sequence comprised of a TATA-box and other sequences that serve to
specify the site of transcription initiation, to which regulatory
elements are added for control of expression. A promoter may be
inducible. Several inducible promoters have been reported (Current
Opinion in Biotechnology, 7:168 (1996)). Examples include the
tetracycline repressor system, Lac repressor system, copper-inducible
systems, salicylate-inducible systems (such as the PR1a system). Also
included are the benzene sulphonamide--(U.S. Pat. No. 5,364,780,
incorporated by reference herein) and alcohol--(WO 97/06269 and WO
97/06268, both incorporated by reference herein) inducible systems and
glutathione S-transferase promoters. In the case of a multicellular
organism, the promoter can also be specific to a particular tissue or
organ or stage of development.

[0160] An enhancer is a DNA sequence which can stimulate promoter activity
and may be an innate element of the promoter or a heterologous element
inserted to enhance the level or tissue specificity of a promoter. It is
capable of operating in both orientations (normal or flipped), and is
capable of functioning even when moved either upstream or downstream from
the promoter. Both enhancers and other upstream promoter elements bind
sequence-specific DNA-binding proteins that mediate their effects.

[0161] The expression cassette can contain a 5' non-coding sequence which
is a nucleotide sequence located 5' (upstream) to the coding sequence. It
is present in the fully processed mRNA upstream of the initiation codon
and may affect processing of the primary transcript to mRNA, stability of
the mRNA, or translation efficiency (Turner et al., Molecular
Biotechnology, 3:225 (1995)).

[0162] The expression cassette may also contain a 3' non-coding sequence,
which is a nucleotide sequence, located 3' (downstream) to a coding
sequence and includes polyadenylation signal sequences and other
sequences encoding regulatory signals capable of affecting mRNA
processing or gene expression. The polyadenylation signal is usually
characterized by affecting the addition of polyadenylic acid tracts to
the 3' end of the mRNA precursor.

[0163] The invention also provides a construct containing a vector and an
expression cassette. The vector may be selected from, but not limited to,
any vector previously described. Into this vector may be inserted an
expression cassette through methods known in the art and previously
described (Sambrook et al., 1989). In one embodiment, the regulatory
sequences of the expression cassette may be derived from a source other
than the vector into which the expression cassette is inserted. In
another embodiment, a construct containing a vector and an expression
cassette is formed upon insertion of a nucleic acid segment of the
invention into a vector that itself contains regulatory sequences. Thus,
an expression cassette is formed upon insertion of the nucleic acid
segment into the vector. Vectors containing regulatory sequences are
available commercially and methods for their use are known in the art
(Clonetech, Promega, Stratagene).

[0164] The expression cassette, or a vector construct containing the
expression cassette may be inserted into a cell. The expression cassette
or vector construct may be carried episomal or integrated into the genome
of the cell.

[0165] A variety of techniques are available and known to those skilled in
the art for introduction of constructs into a cellular host.
Transformation of bacteria and many eukaryotic cells may be accomplished
through use of polyethylene glycol, calcium chloride, viral infection,
phage infection, electroporation and other methods known in the art.
Other transformation methods are available to those skilled in the art,
such as direct uptake of foreign DNA constructs (see EP 295959,
incorporated by reference herein), techniques of electroporation or high
velocity ballistic bombardment with metal particles coated with the
nucleic acid constructs (U.S. Pat. No. 4,945,050, incorporated by
reference herein).

[0166] The selection of an appropriate expression vector will depend upon
the method of introducing the expression vector into host cells.
Typically an expression vector contains (1) prokaryotic DNA elements
coding for a bacterial origin of replication and an antibiotic resistance
gene to provide for the amplification and selection of the expression
vector in a bacterial host; (2) DNA elements that control initiation of
transcription, such as a promoter; (3) DNA elements that control the
processing of transcripts, such as introns, transcription
termination/polyadenylation sequence; and (4) a reporter gene that is
operatively linked to the DNA elements to control transcription
initiation. Useful reporter genes include β-galactosidase,
chloramphenicol acetyl transferase; luciferase, green fluorescent protein
(GFP) and the like.

Methods of Making and Folding

[0167] Galactosyltransferase enzymes of the invention may be produced in
soluble form. Methods that may be used to produce such soluble enzymes
have been described (U.S. Pat. No. 5,032,519, incorporated by reference
in its entirety herein). Briefly, a hydrophobic transmembrane anchor
region of a galactosyltransferase is removed to produce an enzyme that is
in soluble form.

[0169] Alternatively, alpha 1,3 GalNAcT enzymes of the invention may be
produced such that they are anchored in the membrane of a cell. Such
enzymes may be produced that are anchored in the membranes of prokaryotic
and eukaryotic cells. Methods to produce such enzymes have been described
(U.S. Pat. No. 6,284,493, incorporated by reference in its entirety
herein).

[0170] Briefly, in the case of procaryotes, the signal and transmembrane
sequences of the transferase, for example the alpha 1,3 GalNAcT enzyme of
the invention, are replaced by a bacterial signal sequence, capable of
effecting localization of the fusion protein to the outer membrane.
Suitable signal sequences include, but are not limited to those from the
major E. coli lipoprotein Lpp and lam B. In addition, membrane spanning
regions from Omp A, Omp C, Omp F or Pho E can be used in a tripartite
fusion protein to direct proper insertion of the fusion protein into the
outer membrane. Any procaryotic cells can be used in accordance with the
present invention including but not limited to E. coli, Bacillus sp., and
Pseudomonas sp. as representative examples.

[0171] It is also possible, in certain embodiments, that the native
transmembrane domain of the glycosyltransferase, for example the
engineered GalNAcT of the invention as described herein, is replaced by
the transmembrane domain of a bacterial outer membrane protein. In this
embodiment, the alpha 1,3 GalNAcT signal sequence and the bacterial
transmembrane region act in concert to anchor the galactosyltransferase
to the bacterial outer cell membrane. Nearly any outer membrane bound
protein is suitable for this use including but not limited to Omp A, Omp
C, and Omp F, Lpp, and Lam B. The catalytic portion of the GalNAcT should
be fused to an extracellular loop in the bacterial transmembrane region
in order to insure proper orientation of the fusion protein on the outer
membrane surface and not in the cytoplasm or periplasm of the cell.
Insertion of a protein into such a loop region has been previously
reported (Charbit et al., J. Bacteriology, 173:262 (1991); Francisco et
al., Proc. Natl. Acad. Sci., 89:2713 (1992)).

[0173] In another embodiment of the present invention, the transmembrane
domain of the alpha 1,3 GalNAcT can be replaced by the transmembrane
domain of a plasma membrane protein. The transmembrane domain of any
resident plasma membrane protein will be appropriate for this purpose.
The transmembrane portions of the M6 P/IGF-II receptor, LDL receptor or
the transferrin receptor are representative examples.

[0174] In another embodiment the Golgi retention signal of the alpha 1,3
GalNAcT is disrupted by site-directed mutagenesis. This approach mutates
the amino acids responsible for localizing the galactosyltransferase to
the Golgi compartment. The resultant glycosyltransferase is transported
to the plasma membrane where it becomes anchored via its modified
transmembrane sequences.

[0175] In vitro folding of alpha 1,3 GalNAcT requires proper disulfide
bond formation. Ways to ensure proper disulfide bond formation include
S-sulfonation of the protein prior to disulfide formation, use of
oxido-shuffling reagents, and mutation of free Cys residue to Thr. In the
in vitro folding of alpha 3GalNAc-T, the stem region acts as a chaperone.
Additionally, there are additives that can be used to prevent the
hydrophobic collapse, including polyethylene glycol (PEG, e.g. PEG-4000)
or L-arginine-HCl. PEG-4000 and L-arginine are thought to beneficially
affect the solubility of folding intermediates of both catalytic
domain-proteins (CD-proteins) and stem region/catalytic domain proteins
(SRCD-proteins) during in vitro folding or protein obtained from
inclusion bodies. In the case of catalytic domain (CD)-proteins, the
majority of misfolded proteins are insoluble in the absence of PEG-4000
and L-arginine and so they precipitate out during dialysis. Thus, the
process will leave behind the properly folded molecules in solution bound
to UDP-agarose that are enzymatically active.

[0176] When the catalytic domain of alpha 3GalNAc-T is expressed in E.
Coli, it forms insoluble inclusion bodies. These inclusion bodies can be
collected and then solubilized and folded in vitro to produce
catalytically active domains. General methods for isolating and folding
inclusion bodies containing galactosyltransferase catalytic domains have
been previously described (Ramakrishnan et al., J. Biol. Chem., 276:37665
(2001)). Thus, the in vitro folding efficiency is directly related to the
quantity of active enzyme that is produced from the isolated inclusion
bodies. Accordingly, methods to increase the in vitro folding efficiency
would provide increased production of catalytic domains that can be used
to create useful products. US Application 20060084162, incorporated by
reference in its entirety herein, provides materials and methods that
improve in vitro folding of catalytic domains from galactosyltransferases
that are related to the use of a stem region of alpha 3GalNAc-T. Such
methods are of use in the instant invention.

Methods of the Invention

[0177] The methods as described herein provide the ability to conjugate
multiple agents to compounds or compositions of the invention. An
embodiment of the present invention provides a glycoconjugate in which
one or more bioactive agents are bound to a modified saccharide residue,
e.g., a modified galactose, which is in turn bound to a targeting
compound, e.g., a compound capable of binding a receptor on a cell
membrane. The 2' modified galactose can be used as a handle to deliver
therapeutic agents to specific tissue sites. In this manner, many
targeting glycoconjugates can be constructed. For example, a gene
delivery system for genetic therapy can be produced by binding a
nucleotide and a ligand or antibody to the modified sugar. A therapeutic
compound for cancer can be produced by binding a chemotherapeutic agent
and a ligand or antibody, e.g., an antibody to a cancer antigen, to the
modified sugar residue.

[0178] The glycoconjugates can be manufactured as designer
glycoconjugates, according to therapeutic need. As such, the designer
polypeptide itself can be used for the targeting and drug delivery. The
glycoconjugates can be manufactured as nanoparticles. In certain
examples, a biological substrate, such as a bioactive agent, for example
a therapeutic agent, is used to engineer the nanoparticle. In other
examples a second, third, fourth or more bioactive polypeptide is used in
association with the nanoparticle to engineer multivalent nanoparticles.
The bioactive agents do not have to be the same, for example a
nanoparticle comprising three bioactive agents may comprise a
chemotherapeutic, a tracking agent and a targeted delivery agent, such as
an antibody.

[0179] Nanoparticles of the invention have use in methods of treating
diseases. In other examples, the methods of the invention are used to
engineer a glycoprotein from a magnetic resonance agent for use in
diagnostic therapies. In these preferred examples, nanoparticles are
engineered as described herein, where the nanoparticles are
superparamagnetic nanoparticle.

[0180] Polypeptide fragments of the invention having altered donor and
acceptor specificity can be used to catalyze the linkage of numerous
sugars from a donor to numerous acceptor sugars. Linkage of sugar
derivatives can also achieved through use of the altered catalytic
domains of the invention due to their expanded donor and acceptor
specificity.

[0181] The presence of modified sugar moieties on a glycoprotein makes it
possible to link bioactive molecules via modified glycan chains, thereby
assisting in the assembly of bionanoparticles that are useful for
developing the targeted drug delivery system and contrast agents for
example for use in imaging, e.g. magnetic resonance imaging. The
reengineered recombinant glycosyltransferases as described herein also
make it possible to remodel the oligosaccharide chains of glycoprotein
drugs, and to synthesize oligosaccharides for vaccine development.

Targeted Glycoconjugates

[0182] The alpha 1-3 N-Acetylgalactosaminyltransferases (alpha 3 GalNAc-T)
as described herein transfer a sugar from a sugar donor to a sugar
acceptor. A sugar acceptor can be selected from galactose beta 1,4 glcNac
or galactose beta 1,4 glucose. Sugars that can be transferred include
UDP-galactose, UDP-- galactose analogues, UDP-GalNAc and UDP-GalNAc
analogues. This reaction allows galactose to be linked to a sugar
acceptor, for example galactose beta 1,4 glcNAc or galactose beta 1,4
glucose, that may itself be linked to a variety of other molecules, such
as sugars and proteins, e.g., therapeutic agents, imaging agents,
antibodies.

[0183] As described herein, modifications in sugar donors are tolerated by
the alpha 3GalNAc enzymes. The alpha 3GalNAc enzymes of the invention
have the ability to use unnatural substrates (altered donor specificity)
in sugar transfer reactions due to altered donor specificities. The alpha
3 GalNAc-T enzymes have a wider range of donor specificity, e.g. are able
to tolerate a wider range of donors, due to substitutions in the
sugar-nucleotide binding pocket. For example, in certain embodiments as
described herein, substitutions in bovine a3Gal-T that broadens a3Gal-T
donor specificity and makes the enzyme a3 GalNAc-T.

[0184] In certain examples, the one or more substitutions in the substrate
binding site comprise an amino acid substitution at position 280, 281, or
282 corresponding to bovine alpha 1,3 galactosyltransferase (alpha 3
Gal-T) (SEQ ID NO: 21). In another related embodiment, the one or more
substitutions in the substrate hinge region comprise an amino acid
substitution at position 191 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO: 21). In one embodiment,
the one or more substitutions close to the DXD motif comprise an amino
acid substitution at position 228 corresponding to bovine alpha 1,3
galactosyltransferase (alpha 3 Gal-T) (SEQ ID NO 21). In another
embodiment, wherein a serine (S) is substituted for a histidine (H) at
amino acid position 280, a glycine (G) is substituted for an alanine (A)
at amino acid position 281, or a glycine (G) is substituted for an
alanine at amino acid position 282 of (SEQ ID NO 21). In another
embodiment, a serine (S) or an alanine (A) is substituted for a proline
(P) at amino acid position 191 corresponding to (SEQ ID NO: 21). In still
another embodiment, a glutamine (Q) is replaced with a a methionine (M)
at amino acid position 228 of (SEQ ID NO:21).

[0185] In one embodiment of the invention, the donor sugar is modified so
as to include a functional group at the C2 position of the sugar ring,
preferably a ketone or an azido or a thiol functionality. In another
embodiment, the modified sugar is a galactose which is modified at the C2
position by the addition of ketone functionality.

[0186] WO 2005/051429, incorporated by reference in its entirety herein,
describes methods used to bind a bioactive agent to the modified sugar.
The bioactive compounds may preferably include a functional group which
may be useful, for example, in forming covalent bonds with the sugar
residue, which are not generally critical for the activity of the
bioactive agent. Examples of such functional groups include, for example,
amino(--NH:2), hydroxy(--OH), carboxyl (--COOH), thiol(--SH), phosphate,
phosphinate, ketone group, sulfate and sulfinate groups. If the bioactive
compounds do not contain a useful group, one can be added to the
bioactive compound by, for example, chemical synthetic means. Where
necessary and/or desired, certain moieties on the components may be
protected using blocking groups, as is known in the art, see, e.g., Green
& Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons)(1991).

[0187] Exemplary covalent bonds by which the bioactive compounds may be
associated with the sugar residue include, for example, amide (--CONH--);
thioamide (--CSNH--); ether (ROR', where R and R' may be the same or
different and are other than hydrogen); ester (--COO--); thioester
(--COS--); -0-; --S--; --Sn--, where n is greater than 1, preferably
about 2 to about 8; carbamates; --NH--; --NR--, where R is alkyl, for
example, alkyl of from about 1 to about 4 carbons; urethane; and
substituted imidate; and combinations of two or more of these.

[0188] Covalent bonds between a bioactive agent and a modified sugar
residue may be achieved through the use of molecules that may act, for
example, as spacers to increase the conformational and topographical
flexibility of the compound. Examples of such spacers include, for
example, succinic acid, 1,6-hexanedioic acid, 1,8-octanedioic acid, and
the like, as well as modified amino acids, such as, for example,
6-aminohexanoic acid, 4-aminobutanoic acid, and the like.

[0189] One of skill in the art can easily chose suitable compatible
reactive groups for the bioactive agent and the modified sugar, so as to
generate a covalent bond between the bioactive agent and the modified
sugar. Also, while the glycoconjugates of the invention are generally
described with the targeting agent as the acceptor molecule or structure
onto which a donor molecule (e.g., UDP-galactose) is actively linked
through the action of a catalytic domain of a galactosyltransferase the
bioactive agent can also be an acceptor molecule.

[0190] In certain embodiments, the instant method can be used to monitor
glycosylation, for example the glycosylation of therapeutic glycoproteins
and monoclonal antibodies. The potential of glycosyltransferase enzymes
to produce glycoconjugates carrying sugar moieties with reactive groups
may be a benefit to the glycotargeting of drugs to their site of action.
Although a great number of pharmaceutical agents are discovered each
year, the clinical application of these is many times hindered because of
failure to reach the site of action. The methods described herein that
include using reengineered glycosyltransferases to transfer chemically
reactive sugar residues for linking of other molecules via specific
glycan chains may be used as an efficient drug delivery system.

[0191] Detection

[0192] The a3 GalNAc-T as described herein have application in the
detection of specific sugar residues on a glycan chain of a
glycoconjugates and in the glycoconjugation and assembly of
bio-nanoparticles for the targeted delivery of bioactive agents. Protein
glycoslation is one of the most abundant posttranslational modifications
and plays a fundamental role in the control of biological systems and in
disease.

[0193] Accordingly, glycosylation has been found to be a marker in
disease. Additionally, carbohydrate modifications have been shown to be
important for host-pathogen interactions, inflammation, development, and
malignancy (Varki, 1993; Lasky, 1996).

[0194] The methods described herein offer the advantages the modification
occurs in a site directed manner, only where the carbohydrate is attached
to the glycoprotein. Such specificity permits, for example, the use of
site-directed immunotherapy without affecting the antigen binding
affinity of the immunoglobulin. Such specificity permits, further, the
potential use of this approach in developing a drug delivery system or
biological probes.

[0195] Imaging

[0196] Included in the invention are methods for imaging a target cell or
tissue in a subject. The methods as described herein comprise
administering to a subject a polypeptide fragment synthesized by the
method comprising incubating a reaction mixture comprising a polypeptide
fragment from a a3 GalNAc-T with a sugar donor, wherein one or more
imaging agents are linked to the sugar donor, and an sugar acceptor
thereby imaging a target cell or tissue.

[0197] Coupling

[0198] Methods of transfer of C2 modified galactose analogues, for example
C2 keto galactose from its UDP derivative to the GlcNAc residue on the
N-glycan chain of ovalbumin or to an asialo-agalacto-IgG1 molecule have
been described in the art, for example in WO 2005/051429, incorporated by
reference in its entirety herein. The C2 modified galactose analogues,
for example C2 keto galactose can be biotinylated, thus allowing for
biotinylation of carriers such as ovalbumin and IgG.

[0199] The method of coupling a target agent to a carrier protein via
glycan chains, for example ovalbumin and IgG1, is advantageous over other
cross-linking methods. In the instant method, the target agent is linked
in a site-directed manner, only where the carbohydrate is attached to the
glycoprotein, for example as in the IgG1 molecule at the Fc domain, away
from the antigen binding site. A problem encountered in previous
approaches using monoclonal antibodies for immunotherapy is the lack of
specificity of the reactions, resulting in heterologous labeling and a
decrease in the antibody affinity for the antigen. The instant invention
overcomes this problem.

[0200] Accordingly, the invention features methods of coupling an agent or
agents to a carrier protein. The methods described herein comprise
coupling an agent to a carrier protein comprising incubating a reaction
mixture comprising a polypeptide fragment of an alpha 1,3
N-Acetylgalactosylaminotransferase (alpha 3GalNaCT) that retains the
ability to transfer a sugar with a chemically reactive functional group
from a sugar donor to a sugar acceptor, wherein the sugar donor is
coupled to an agent and the sugar acceptor is a carrier protein.

[0202] The sugar donor is, in certain examples, a UDP-galactose analogue,
that can comprise an azido group, a keto group, or a thiol group. The
azido group, the keto group or the thiol group can be substituted at the
C2 position of galactose, thus allowing for linking of agents.
Accordingly, in certain preferred examples, one or more agents are linked
to a sugar moiety of the sugar donor. The agent can be selected from the
group consisting of: antibodies, single chain antibodies, bacterial
toxins, growth factors, therapeutic agents, targeting agents, contrast
agents, chemical labels, a radiolabels, and fluorescent labels.

[0203] The carrier protein, in preferred examples, is ovalbumin. The
carrier protein, in other preferred examples, is an IgG. In certain
instances, it is advantageous to couple the C2 UDP-galactose analogue to
biotin for detection. Subsequent detection of biotin can be carried out
by chemiluminescent assay. The method as described herein is useful for
imaging procedures, for example in magnetic resonance imaging.

Carbohydrate Synthesis

[0204] Enzymatic carbohydrate synthesis using glycosyltransferases is
regio- and stereospecific and does not require extensive protecting group
designs. Naturally occurring carbohydrates have been successfully
prepared by this biomimetic pathway. The novel
alpha(1-3)galactosylaminotransferases described herein have use in, for
example, the design and synthesis of natural and non-natural carbohydrate
libraries for pharmaceutical purposes, for example, the synthesis of
sialyl-Lewis(a)- and sialyl-Lewis(x)-libraries. Baisch et al. (Carbohydr
Res. 1998 November; 312(1-2):61-72, incorporated by reference in its
entirety herein).

[0205] Mimetics of a terminal tetrasaccharide region of many cellular
glycoproteins and glycolipids (sialyl Lewis X) have demonstrated to
inhibit angiogenesis both in vitro and in vivo and this may be used for
cancer treatment

Antibodies and Applications

[0206] As described herein, the targeting compound may be an antibody or a
fragment thereof. The term"antibody" (Ab) or"monoclonal antibody" (Mab)
is meant to include intact molecules as well as antibody portions (e.g.,
Fab and F (ab')2 portions and Fv fragments) which are capable of
specifically binding to a cell surface marker. Such portions are
typically produced by proteolytic cleavage, using enzymes such as papain
(to produce Fab portions) or pepsin (to produce F (ab')2 portions).
Alternatively, antigen-binding portions can be produced through the
application of recombinant DNA technology.

[0207] The immunoglobulin can be a "chimeric antibody" as that term is
recognized in the art. Also, the immunoglobulin may be a bifunction or a
hybrid antibody, that is, an antibody which may have one arm having a
specificity for one antigenic site, such as a tumor associated antigen,
while the other arm recognizes a different target, for example, a hapten
which is, or to which is bound, an agent lethal to the antigen-bearing
tumor cell. Alternatively, the bifunctional antibody may be one in which
each arm has specificity for a different epitope of a tumor associated
antigen of the cell to be therapeutically or biologically modified. In
any case, the hybrid antibodies have a dual specificity, preferably with
one or more binding sites specific for the hapten of choice or one or
more binding sites specific for a target antigen, for example, an antigen
associated with a tumor, an infectious organism, or other disease state.

[0208] Biological bifunctional antibodies are described, for example, in
European Patent Publication, EPA 0 105 360, which is incorporated herein
by reference. Hybrid or bifunctional antibodies may be derived
biologically, by cell fusion techniques, or chemically, especially with
cross-linking agents or disulfide bridge-forming reagents, and may be
comprised of those antibodies and/or fragments thereof. Methods for
obtaining such hybrid antibodies are disclosed, for example, in PCT
application WO83/03679, published Oct. 27, 1983, and published European
Application EPA 0 217 577, published Apr. 8, 1987, which are incorporated
herein by reference. In one embodiment, the bifunctional antibodies are
biologically prepared from a polydome or a quadroma, or are synthetically
prepared with cross-linking agents such as bis-(maleimideo)-methyl
ether("BMME"), or with other cross-linking agents familiar to those
skilled in the art.

[0210] The antibodies may, in certain embodiments, be chimeric monoclonal
antibodies. As used herein, the term "chimeric antibody" refers to a
monoclonal antibody comprising a variable region, i.e., binding region,
from one source or species and at least a portion of a constant region
derived from a different source or species, usually prepared by
recombinant DNA techniques.

[0211] Chimeric antibodies comprising a murine variable region and a human
constant region are preferred in certain applications of the invention,
particularly human therapy, because such antibodies are readily prepared
and may be less immunogenic than purely murine monoclonal antibodies.
Such murine/human chimeric antibodies are the product of expressed
immunoglobulin genes comprising DNA segments encoding murine
immunoglobulin variable regions and DNA segments encoding human
immunoglobulin constant regions. Other forms of chimeric antibodies
encompassed by the invention are those in which the class or subclass has
been modified or changed from that of the original antibody. Such
"chimeric" antibodies are also referred to as "class-switched
antibodies." Methods for producing chimeric antibodies involve
conventional recombinant DNA and genetransfection techniques well known
in the art. See, e.g., Morrison, S. L. et al., Proc. Nat'l Acad. Sci.,
81: 6851 (1984).

[0212] Encompassed by the term "chimeric antibody" is the concept of
"humanized antibody," that is those antibodies in which the framework or
"complementarity" determining regions ("CDR") have been modified to
comprise the CDR of an immunoglobulin of different specificity as
compared to that of the parent immunoglobulin. (See, e.g., EPA 0 239 400
(published Sep. 30, 1987)) In a preferred embodiment, a murine CDR is
grafted into the framework region of a human antibody to prepare the
"humanized antibody." See, e.g., L. Riechmann et al., Nature, 332: 323
(1988); M. S, Neuberger et al., Nature, 314: 268 (1985). Furthermore, the
immunoglobulin (antibody), or fragment thereof, used in the present
invention may be polyclonal or monoclonal in nature. Monoclonal
antibodies are the preferred immunoglobulins. The preparation of such
polyclonal or monoclonal antibodies is well known to those skilled in the
art. See, e.g., G. Kohler and C. Milstein, Nature, 256: 495 (1975). The
antibodies of the present invention may be prepared by any of a variety
of methods. For example, cells expressing the cell surface marker or an
antigenic portion thereof can be administered to an animal in order to
induce the production of sera containing polyclonal antibodies. In a
preferred method, a preparation of protein is prepared and purified so as
to render it substantially free of natural contaminants. Such a
preparation is then introduced into an animal in order to produce
polyclonal antisera of greater specific activity. However, the present
invention should not be construed as limited in scope by any particular
method of production of an antibody whether bifunctional, chimeric,
bifunctional-chimeric, humanized, or an antigen-recognizing fragment or
derivative thereof.

[0213] In a preferred embodiment, the antibodies of the present invention
are monoclonal antibodies (or portions thereof). Such monoclonal
antibodies can be prepared using hybridoma technology (Kohler et al.,
Nature, 256: 495 (1975); Kohler et al., Eur. J. Immunol., 6: 511 (1976);
Kohler et al, Eur. J. Immunol., 6: 292 (1976); Hammerling et al., In:
"Monoclonal Antibodies and T-Cell Hybridomas," Elsevier, N.Y., pp.
563-681 (1981)). In general, such procedures involve immunizing an animal
(preferably a mouse) with a protein antigen or with a protein-expressing
cell (suitable cells can be recognized by their capacity to bind
antibody). The splenocytes of such immunized mice are extracted and fused
with a suitable myeloma cell line. Any suitable myeloma cell line may be
employed in accordance with the present invention. After fusion, the
resulting hybridoma cells are selectively maintained in HAT medium, and
then cloned by limiting dilution as described by Wands et al.,
Gastroenterology, 80: 225-232 (1981). The hybridoma cells obtained
through such a selection are then assayed to identify clones which
secrete antibodies capable of binding the antigen. In addition,
hybridomas and/or monoclonal antibodies which are produced by such
hybridomas and which are useful in the practice of the present invention
are publicly available from sources such as the American Type Culture
Collection or commercial retailers.

[0215] In one embodiment, the glycoconjugates of the invention include
monoclonal antibodies, such as those directed against tumor antigens, for
use as cancer therapeutics. Generally, monoclonal antibodies have one
N-linked bi-antennary oligosaccharide attached at the IgG-Fc region. The
terminal sugars of the oligosaccharide moiety come in several glycoforms,
for example, some are desolated, degalactosylated, with only terminal
N-acetylglucosaminyl residues.

[0216] The monoclonal antibodies carrying only terminal
N-acetylglucosamine on the bi-antennary oligosaccharide moieties, the
Goglycoform, can be generated by de-sialylation and de-galactosylation of
the monoclonal antibodies. With the Tyr289Leu-Gal-TI (Y289LGaITI) enzyme
and UDP-a-galactose-C-2-modified, a galactose moiety that has a
chemically reactive group attached at the C2 position of galactose, can
then be transferred to Go glycoform of the monoclonal antibody. The
chemically reactive group can include, for example, a ketone moiety that
can serve as a neutral, yet versatile chemical handle to add other
agents, such as bioactive agents, to the compound.

Methods of Treatment

[0217] The instant invention provides enzymes and methods that can be used
to promote the chemical linkage of biologically important molecules that
have previously been difficult to link, and thus provides a means to link
agents for therapeutic application. Moreover, the instant invention
provides a means to carry out the method in a physiological setting.

[0218] Accordingly, the invention features methods for the diagnosis or
treatment of a subject suffering from a disease or disorder. The methods
comprise administering to the subject an effective amount of polypeptide
fragment synthesized by the method comprising incubating a reaction
mixture comprising an isolated catalytic domain from an alpha 1,3
N-Acetylgalactosaminyltransferase (alpha3GalNac-T) with a sugar donor,
wherein one or more agents are linked to the sugar donor, and an sugar
acceptor thereby diagnosing or treating the subject.

[0220] Disease states needing treatment are only limited by current
available therapeutics. As described herein, the methods of the invention
are useful for engineering of nanoparticles, including multivalent
nanoparticles, carrying any number of therapeutic agents. For example,
the nanoparticles can be used to treat cancer, inflammatory disease,
cardiovascular disease, obesity, ageing, bacterial infection, or any
other disease amenable to therapy.

[0221] The glycoconjugates compositions of the invention can be used to
treat and/or diagnose a variety of diseases and/or disorders. For
example, the glycoconjugates compositions of the invention are used for
specific, targeted delivery of bioactive agents, including toxic drugs,
agents for imaging or diagnostics, (e.g., toxins, radionuclides), to
therapeutically-relevant tissues or cells of the body, for example,
tumors. In another embodiment of the invention, the glycoconjugates
compositions of the invention are used to deliver bioactive agents,
including DNA vectors, to cells.

[0222] As further examples, the glycoconjugates compositions of the
invention are useful for the treatment of a number of diseases and/or
disorders including, but not limited to: cancer, both solid tumors as
well as blood-borne cancers, such as leukemia; hyperproliferative
disorders that can be treated by the compounds of the invention include,
but are not limited to, neoplasms located in the: abdomen, bone, breast,
digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal,
parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and
neck, nervous (central and peripheral), lymphatic system, pelvic, skin,
soft tissue, spleen, thoracic, and urogenital.

[0224] The glycoconjugates of the invention can be used to treat genetic
diseases, such as enzyme deficiency diseases.

[0225] The glycoconjugates of the invention can be used to treat
hyperproliferative disorders. Examples of such hyperproliferative
disorders that can be treated by the glycoconjugates of the invention are
as described in Application WO 2005/051429, and are incorporated by
reference in its entirety herein.

[0226] The glycoconjugates of the present invention are also useful for
raising an immune response against infectious agents. Viruses are one
example of an infectious agent that can cause disease or symptoms that
can be treated by the compounds of the invention. Examples of viruses
that can cause disease or symptoms and that can be treated by the
glycoconjugates of the invention are as described in Application WO
2005/051429, and are incorporated by reference in its entirety herein.

[0227] Similarly, bacterial or fungal agents that can cause disease or
symptoms and that can be treated by the glycoconjugates of the invention
are as described in Application WO 2005/051429, and are incorporated by
reference in its entirety herein.

[0228] Additionally, the glycoconjugates of the invention are useful for
treating autoimmune diseases. An autoimmune disease is characterized by
the attack by the immune system on the tissues of the victim. Autoimmune
disease is characterized by the inability of the recognition of "self"
and the tissue of the afflicted subject is treated as a foreign target.
The compounds of the present invention are therefore useful for treating
autoimmune diseases by desensitizing the immune system to these self
antigens by provided a TCR signal to T cells without a costimulatory
signal or with an inhibitory signal. Examples of autoimmune diseases
which may be treated using the glycoconjugates of the present invention
are as described in Application WO 2005/051429, and are incorporated by
reference in its entirety herein.

[0229] Similarly, allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may also be
treated by glycoconjugates of the invention. Moreover, the
glycoconjugates of the invention can be used to treat anaphylaxis,
hypersensitivity to an antigenic molecule, or blood group
incompatibility.

[0231] The invention also provides methods for eliciting an immune
response in a mammal such as a human, including administering to a
subject an immunological composition comprising a compound or composition
as described herein. Therefore, one embodiment of the present invention
is to use the glycoconjugates described herein in an immunological
preparation.

[0232] The immunological composition according to the instant invention
may be prepared by any method known in the art. For example,
glycoconjugates of the present invention are prepared and are then
injected into an appropriate animal. The compositions according to the
present invention may be administered in a single dose or they may be
administered in multiple doses, spaced over a suitable time scale to
fully utilize the secondary immunization response. For example, antibody
titers may be maintained by administering boosters once a month. The
vaccine may further comprise a pharmaceutically acceptable adjuvant,
including, but not limited to Freund's complete adjuvant, Freund's
incomplete adjuvant, lipopolysaccharide, monophosphoryl lipid A, muramyl
dipeptide, liposomes containing lipid A, alum, muramyl
tripeptide-phosphatidylethanolamine, keyhole and limpet hemocyanin.

Administration

[0233] The compositions of the present invention may be administered by
any means that results in the contact of the bioactive agent with the
agent's site or site (s) of action on or in a subject, e.g., a patient.
The compositions may be administered alone or in conjunction with one or
more other therapies or treatments.

[0234] The targeted glycoconjugates produced according to the present
invention, can be administered to a mammalian host by any route. Thus, as
appropriate, administration can be orally, intravenously, rectally,
parenterally, intracistemally, intradermally, intravaginally,
intraperitoneally, topically (as by powders, ointments, gels, creams,
drops or transdermal patch), bucally, or as an oral or nasal spray. The
term "parenteral" as used herein refers to modes of administration which
include intravenous, intramuscular, intraperitoneal, intrasternal,
subcutaneous and intraarticular injection and infusion. Parenteral
administration in this respect includes administration by the following
routes: intravenous, intramuscular, subcutaneous, intraocular,
intrasynovial, transepithelial including transdermal, ophthalmic,
sublingual and buccal; topically including ophthalmic, dermal, ocular,
rectal and nasal inhalation via insufflation, aerosol and rectal
systemic.

[0235] In addition, administration can be by periodic injections of a
bolus of the therapeutic or can be made more continuous by intravenous or
intraperitoneal administration from an external source. In certain
embodiments, the therapeutics of the instant invention can be
pharmaceutical-grade and incompliant with the standards of purity and
quality control required for administration to humans. Veterinary
applications are also within the intended meaning as used herein.

[0236] The formulations, both for veterinary and for human medical use, of
the therapeutics according to the present invention typically include
such therapeutics in association with a pharmaceutically acceptable
carrier therefor and optionally other ingredient (s). The carrier (s) can
be acceptable in the sense of being compatible with the other ingredients
of the formulations and not deleterious to the recipient thereof.
Pharmaceutically acceptable carriers are intended to include any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration.

[0237] A pharmaceutical composition of the invention is formulated to be
compatible with its intended route of administration. Solutions or
suspensions used for parenteral, intradermal, or subcutaneous application
can include the following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols, glycerine,
propylene glycol or other synthetic solvents; antibacterial agents such
as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid
or sodium bisulfite; chelating agents such asethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents for the
adjustment of tonicity such as sodium chloride or dextrose. pH can be
adjusted with acids or bases, such as hydrochloric acid or sodium
hydroxide.

[0238] Useful solutions for oral or parenteral administration can be
prepared by any of the methods well known in the pharmaceutical art,
described, for example, in Remington's Pharmaceutical Sciences.
Formulations for parenteral administration also can include glycocholate
for buccal administration, methoxysalicylate for rectal administration,
or citric acid for vaginal administration. The parenteral preparation can
be enclosed in ampoules, disposable syringes or multiple dose vials made
of glass or plastic.

[0239] Formulations of the present invention suitable for oral
administration can be in the form of discrete units such as capsules,
gelatin capsules, sachets, tablets, troches, or lozenges, each containing
a predetermined amount of the drug; in the form of a powder or granules;
in the form of a solution or a suspension in an aqueous liquid or
non-aqueous liquid; or in the form of an oil-in-water emulsion or a
water-in-oil emulsion. The therapeutic can also be administered in the
form of a bolus, electuary or paste. A tablet can be made by compressing
or molding the drug optionally with one or more accessory ingredients.
Compressed tablets can be prepared by compressing, in a suitable machine,
the drug in a free-flowing form such as a powder or granules, optionally
mixed by a binder, lubricant, inert diluent, surface active or dispersing
agent. Molded tablets can be made by molding, in a suitable machine, a
mixture of the powdered drug and suitable carrier moistened with an inert
liquid diluent.

[0240] Oral compositions generally include an inert diluent or an edible
carrier.

[0241] For the purpose of oral therapeutic administration, the active
compound can be incorporated with excipients. Oral compositions prepared
using a fluid carrier for use as a mouthwash include the compound in the
fluid carrier and are applied orally and swished and expectorated or
swallowed. Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets, pills,
capsules, troches and the like can contain any of the following
ingredients, or compounds of a similar nature: a binder such as
microcrystalline cellulose, gumtragacanth or gelatin; an excipient such
as starch or lactose; a disintegrating agent such as alginic acid,
Primogel, or corn starch; a lubricant such as magnesium stearate or
Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent
such as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.

[0242] Pharmaceutical compositions suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. For intravenous administration, suitable
carriers include physiological saline, bacteriostatic water, Cremophor
ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all
cases, the composition can be sterile and can be fluid to the extent that
easy syringability exists. It can be stable under the conditions of
manufacture and storage and can be preserved against the contaminating
action of microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. The proper fluidity
can be maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and antifungal
agents, for example, parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars, polyalcohols such as manitol,
sorbitol, and sodium chloride in the composition. Prolonged absorption of
the injectable compositions can be brought about by including in the
composition an agent which delays absorption, for example, aluminum
monostearate and gelatin.

[0243] Sterile injectable solutions can be prepared by incorporating the
active compound in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required, followed
by sterilization, e.g., filtered sterilization. Generally, dispersions
are prepared by incorporating the active compound into a sterile vehicle
which contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile powders
for the preparation of sterile injectable solutions, methods of
preparation include vacuum drying and freeze-drying which yields a powder
of the active ingredient plus any additional desired ingredient.

[0244] Formulations suitable for topical administration, including eye
treatment, include liquid or semi-liquid preparations such as liniments,
lotions, gels, applicants, oil-in-water or water-in-oil emulsions such as
creams, ointments or pasts; or solutions or suspensions such as drops.
Formulations for topical administration to the skin surface can be
prepared by dispersing the therapeutic with a dermatologically acceptable
carrier such as a lotion, cream, ointment or soap. In some embodiments,
useful are carriers capable of forming a film or layer over the skin to
localize application and inhibit removal.

[0245] For inhalation treatments, such as for asthma, inhalation of powder
(self-propelling or spray formulations) dispensed with a spray can, a
nebulizer, or an atomizer can be used. Such formulations can be in the
form of a finely comminuted powder for pulmonary administration from a
powder inhalation device or self-propelling powder-dispensing
formulations. In the case of self-propelling solution and spray
formulations, the effect can be achieved either by choice of a valve
having the desired spray characteristics (i.e., being capable of
producing a spray having the desired particle size) or by incorporating
the active ingredient as a suspended powder in controlled particle size.
For administration by inhalation, the therapeutics also can be delivered
in the form of an aerosol spray from a pressured container or dispenser
which contains a suitable propellant, e.g., a gas such as carbon dioxide,
or a nebulizer. Nasal drops also can be used.

[0246] Systemic administration also can be by transmucosal ortransdermal
means. For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the formulation.
Such penetrants generally are known in the art, and include, for example,
for transmucosal administration, detergents, bile salts, and filsidic
acid derivatives. Transmucosal administration can be accomplished through
the use of nasal sprays or suppositories. Fortransdermal administration,
the therapeutics typically are formulated into ointments, salves, gels,
or creams as generally known in the art.

[0247] The therapeutics can be prepared with carriers that will protect
against rapid elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery systems.

[0249] The compositions can be formulated in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form refers to
physically discrete units suited as unitary dosages for the subject to be
treated; each unit containing a predetermined quantity of active compound
calculated to produce the desired therapeutic effect in association with
the required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in the
art of compounding such an active compound for the treatment of
individuals.

[0250] Generally, the therapeutics identified according to the invention
can be formulated for administration to humans or other mammals, for
example, in therapeutically effective amounts, e.g., amounts which
provide appropriate concentrations of the bioactive agent to target
tissue/cells for a time sufficient to induce the desired effect.
Additionally, the therapeutics of the present invention can be
administered alone or in combination with other molecules known to have a
beneficial effect on the particular disease or indication of interest. By
way of example only, useful cofactors include symptom-alleviating
cofactors, including antiseptics, antibiotics, antiviral and antifungal
agents and analgesics andanesthetics.

[0251] The effective concentration of the therapeutics identified
according to the invention that is to be delivered in a therapeutic
composition will vary depending upon a number of factors, including the
final desired dosage of the drug to be administered and the route of
administration. The preferred dosage to be administered also is likely to
depend on such variables as the type and degree of the response to be
achieved; the specific composition of another agent, if any, employed;
the age, body weight, general health, sex and diet of the patient; the
time of administration, route of administration, and rate of excretion of
the composition; the duration of the treatment; bioactive agent (such as
a chemotherapeutic agent) used in combination or coincidental with the
specific composition; and like factors well known in the medical arts. In
some embodiments, the therapeutics of this invention can be provided to
an individual using typical dose units deduced from the earlier-described
mammalian studies using non-human primates and rodents. As described
above, a dosage unit refers to a unitary, i.e. a single dose which is
capable of being administered to a patient, and which can be readily
handled and packed, remaining as a physically and biologically stable
unit dose comprising either the therapeutic as such or a mixture of it
with solid or liquid pharmaceutical diluents or carriers.

[0252] Therapeutics of the invention also include "prodrug" derivatives.
The term prodrug refers to a pharmacologically inactive (or partially
inactive) derivative of a parent molecule that requires
biotransformation, either spontaneous or enzymatic, within the organism
to release or activate the active component. Prodrugs are variations or
derivatives of the therapeutics of the invention which have groups
cleavable under metabolic conditions. Prodrugs become the therapeutics of
the invention which are pharmaceutically active in vivo, when they
undergo solvolysis under physiological conditions or undergo enzymatic
degradation. Prodrug forms often offer advantages of solubility, tissue
compatibility, or delayed release in the mammalian organism (see,
Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985
and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp.
352-401, Academic Press, San Diego, Calif., 1992).

[0254] Application No. WO 2005/051429, incorporated by reference in its
entirety herein, provides a list of exemplary agents that can be
conjugated to the compositions of the instant invention.

Kits

[0255] Also included in the invention are kits. Preferably, kits comprise
a packaging material, and a polypeptide fragment from an alpha 1,3
N-Acetylgalactosaminyltransferase (alpha3GalNac-T) according to any one
of the aspects of the invention as described herein. The kits, in certain
preferred embodiments, comprise a sugar donor. The donor can be any one
of UDP-galactose, UDP-GalNAc, UDP-GalNAc analogues or UDP-Galactose
analogues. The kits can also comprise an agent. In preferred examples,
the agent is linked to the sugar donor. Exemplary agents are described in
this disclosure. Certain agents can be selected from antibodies, single
chain antibodies, bacterial toxins, growth factors, therapeutic agents,
contrast agents, targeting agents, chemical labels, a radiolabels, and
fluorescent labels.

EXAMPLES

[0256] It should be appreciated that the invention should not be construed
to be limited to the examples that are now described; rather, the
invention should be construed to include any and all applications
provided herein and all equivalent variations within the skill of the
ordinary artisan.

[0257] As described in more detail below, the experiments reported herein
are based on the structure-based design of a 1,3
N-Acetylgalactosaminyltransferase (a3 GalNac-T) from a 1,3
galactosyltransferase (a3 Gal-T) which was mutated at seven positions to
broaden a3Gal-T donor specificity and make it a3GalNac-T.

[0258] X-ray crystal structures of the catalytic domain of many
glycosyltransferases have been determined in recent years, and these
studies show that the specificity of the sugar donor is determined by
residues in the sugar-nucleotide binding pocket of glycosyltransferases.
This structural information has made it possible to reengineer the
existing glycosyltransferases.

[0259] Described herein is the stricture based design of an alpha
1-3-N-acetylgalactosaminyltransferase from a1-3-galactosyltransferase.
FIG. 1 is a schematic showing the structure--based design of a
1-3-N-acetylgalactosaminyltransferase from a1-3-galactosyltransferase. In
FIG. 2, the sugar donor binding site and the hinge region where the
substitutions occur are shown. These regions are the regions where the
substitutions occur in the a1-3-galactosyltransferase. Mutation of
residues in these regions leads to the novel alpha 1-3
GalNAc-transferases described herein that can transfer 2'-modified
galactose.

[0260]FIG. 3 and FIG. 4 shows transfer of UDP--modified sugars by the
alpha 1,3 Gal-T-191A. 280SGG282 enzyme. FIG. 5 is a Table showing the
effect of substitutions in the donor substrate binding site, hinge region
and near DXD motif on Gal activity, GalNAc activity and GalKeto activity
is shown.

Methods

[0261] The invention was performed using the following methods:

Met344His Mutant

[0262] Site-directed mutagenesis was performed using the PCR method.
Construction of the enzymes was carried out as described previously in
Qasba et al. (Biochemistry 2004, 43, 12513-12522), incorporated by
reference in its entirety herein.

Bacterial Growth and Plasmid Transformation

[0263] Bacterial growth and plasmid transformations can be performed using
standard procedures (Ausubel et al., Current Protocols in Molecular
Biology, Greene Publishing Associates and Wiley-Interscience, New York
(1987)). US Published Application 20060084162, incorporated by reference
in its entirety herein, describes methods for bacterial growth and
transformation using the plasmid pEGT-dl29, which encodes the catalytic
domain (residues 130-402) of bovine β (1,4)-galactosyltransferase I.
Site-directed mutagenesis can be performed using a CLONTECH site-directed
mutagenesis transformer kit. Thus, the transformation mixture contains
the template pEGT-d129, a selection primer, and a mutagenic primer for
creation of a desired enzyme. Enzymes are screened for the incorporated
substitutions by looking for changes in restriction enzyme digestion
patterns and confirmed by DNA sequencing. The positive clones were
transformed into B834(DE3)pLysS cells.

Expression and Purification of Inclusion Bodies

[0264] The expression and purification of the inclusion bodies can be
carried out as described previously (Ausubel et al., Current Protocols in
Molecular Biology, Greene Publishing Associates and Wiley-Interscience,
New York (1987)). The inclusion bodies are S-sulfonated by dissolving in
5 M GdnHCl, 0.3 M sodium sulfite, and the addition of di-sodium
2-nitro-5-thiosulfobenzoate to a final concentration of 5 mM. The
sulfonated protein is precipitated by dilution with water, and the
precipitate was washed thoroughly.

[0265] Briefly, 100 mg of sulfonated protein is folded in one liter
folding solution for 48 hours. Inclusion of 10% glycerol and 10 mM
lactose in the folding solution enhances the folding efficiency of the
galactosyltransferase, e.g. beta-1,4-galactosyltransferase (beta4Gal-T1).
After refolding the protein, the folding solution is extensively dialyzed
against water. During dialysis the misfolded protein precipitates out,
while the folded protein remains soluble. The soluble protein is first
concentrated and then purified on a Ni-column. Nearly 2 mg of folded
ppGalNAc-T2 protein is obtained form 1 liter of folding solution.
Purified protein may be tested for catalytic activity using a 13 amino
acid peptide, PTTDSTTPAPTTK, as an acceptor using methods described
previously (Fritz. T. A et al. J Biol. Chem. 2006).

[0267] Mutation of certain amino acid residues as described herein is, in
certain examples, performed site-directed mutagenesis. US Published
Application 20060084162 describes methods for site directed mutagenesis
of amino acid position 289 of the bovine β
(1,4)-galactosyltransferase I, performed using the PCR method. The method
is easily adapted by one of skill in the art to the instant invention for
engineering of 1,3 N-Acetylgalactosylaminotransferase (alpha 3GalNaCT)
enzymes from the alpha Gal T.

Gal-T and GalNAc-T Enzyme Assays

[0268] Gal-T and GalNAc T enzyme assays are easily performed according to
methods described in the art, for example US Published Application
20060084162. Protein concentrations are measured using the Bio-Rad
protein assay kit, based on the method of Bradford and further verified
on SDS gel. An in vitro assay procedure for the Gal-T1 has been reported
previously (Ramakrishnan et al., J. Biol. Chem., 270, 87665-376717
(2001)). The activities were measured using UDP-Gal or UDP-GalNAc as
sugar nucleotide donors, and GlcNAc and Glc as the acceptor sugars. For
the specific activity measurements, a 100-μl incubation mixture
containing 50 mM β-benzyl-GlcNAc, 10 mM MnCl2, 10 mM Tris-HCl,
pH 8.0, 500μM UDP-Gal or UDP-GalNAc, 20 ng of Gal-T1, and 0.5μCl of
[3H]UDP-Gal or [3H]UDP-GalNAc was used for each Gal-T or
GalNAc-T reaction. The incubation was carried out at 37° C. for 10
min. The reaction was terminated by adding 200μl of cold 50 mM EDTA,
and the mixture was passed through a 0.5-ml bed volume column of AG1-X8
cation resin (Bio-Rad) to remove any unreacted [3H]UDP-Gal or
[3H]UDP-GalNAc. The column was washed successfully with 300, 400,
and 500μl of water, and the column flow-through was diluted with
Biosafe scintillation fluid; radioactivity was measured with a Beckman
counter. A reaction without the acceptor sugar was used as a control. A
similar assay was carried out to measure the GalNAc-T activity with Glc
and other acceptors in the presence of 50μM bovine LA (Sigma).

[0269] The in vitro assay for enzyme activity (beta Gal T1, double mutant
beta-gal) can be performed as described (Boeggeman et al., Glycobiology,
12:395-407 (2002)). The 3H-labeled-UDP-Gal or UDP-Galactose was used
as sugar donor and GlcNAc as the sugar acceptor. A reaction without
GlcNAc was used as a control.

Other Embodiments

[0270] From the foregoing description, it will be apparent that variations
and modifications may be made to the invention described herein to adopt
it to various usages and conditions. Such embodiments are also within the
scope of the following claims.

[0271] The recitation of a listing of elements in any definition of a
variable herein includes definitions of that variable as any single
element or combination (or subcombination) of listed elements. The
recitation of an embodiment herein includes that embodiment as any single
embodiment or in combination with any other embodiments or portions
thereof.

[0272] All patents and publications mentioned in this specification are
herein incorporated by reference to the same extent as if each
independent patent and publication was specifically and individually
indicated to be incorporated by reference.